Human monoclonal antibodies to ctla-4

ABSTRACT

In accordance with the present invention, there are provided fully human monoclonal antibodies against human cytotoxic T-lymphocyte antigen 4 (CTLA-4). Nucleotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly contiguous heavy and light chain sequences spanning the complementarity determining regions (CDRs), specifically from within FR1 and/or CDR1 through CDR3 and/or within FR4, are provided. Further provided are antibodies having similar binding properties and antibodies (or other antagonists) having similar functionality as antibodies disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 13/399,448, filed Feb. 17, 2012, which is acontinuation to application of U.S. patent application Ser. No.11/981,824, filed Oct. 31, 2007, issued as U.S. Pat. No. 8,143,379 onMar. 27, 2012, which is a continuation application of U.S. patentapplication Ser. No. 11/128,900, filed May 12, 2005, issued as U.S. Pat.No. 7,411,057 on Aug. 12, 2008, which is a continuation application ofU.S. patent application Ser. No. 10/776,649, filed Feb. 10, 2004, issuedas U.S. Pat. No. 7,132,281 on Nov. 7, 2006, which is a divisionalapplication of U.S. patent application Ser. No. 10/612,497, filed Jul.1, 2003, issued as U.S. Pat. No. 7,109,003 on Sep. 19, 2006, which is acontinuation application of U.S. patent application Ser. No. 09/472,087,filed Dec. 23, 1999, issued as U.S. Pat. No. 6,682,736 on Jan. 27, 2004,which claims priority to U.S. Provisional Patent Application No.60/113,647, filed Dec. 23, 1998. The disclosures of all theaforementioned priority applications are incorporated by reference intheir entirety herein.

BACKGROUND OF THE INVENTION Summary of the Invention

In accordance with the present invention, there are provided fully humanmonoclonal antibodies against human cytotoxic T-lymphocyte antigen 4(CTLA-4). Nucleotide sequences encoding and amino acid sequencescomprising heavy and light chain immunoglobulin molecules, particularlycontiguous heavy and light chain sequences spanning the complementaritydetermining regions (CDRs), specifically from within FR1 and/or CDR1through CDR3 and/or within FR4, are provided. Further provided areantibodies having similar binding properties and antibodies (or otherantagonists) having similar functionality as antibodies disclosedherein.

BACKGROUND OF THE TECHNOLOGY

Regulation of immune response in patients would provide a desirabletreatment of many human diseases that could lead to a specificity ofaction that is rarely found through the use of conventional drugs. Bothup-regulation and down-regulation of responses of the immune systemwould be possible. The roles of T cells and B cells have beenextensively studied and characterized in connection with the regulationof immune response. From these studies, the role of T cells appear, inmany cases, to be particularly important in disease prevention andtreatment.

T cells possess very complex systems for controlling their interactions.Interactions between T cells utilize numerous receptors and solublefactors for the process. Thus, what effect any particular signal mayhave on the immune response generally varies and depends on theparticular factors, receptors and counter-receptors that are involved inthe pathway. The pathways for down-regulating responses are as importantas those required for activation. Thymic education leading to T-celltolerance is one mechanism for preventing an immune response to aparticular antigen. Other mechanisms, such as secretion of suppressivecytokines, are also known.

Activation of T cells requires not only stimulation through the antigenreceptor (T cell receptor (TCR)), but additional signaling throughco-stimulatory surface molecules such as CD28. The ligands for CD28 arethe B7-1 (CD80) and B7-2 (CD86) proteins, which are expressed onantigen-presenting cells such as dendritic cells, activated B-cells ormonocytes that interact with T-cell CD28 or CTLA-4 to deliver acostimulatory signal. The role of costimulatory signaling was studied inexperimental allergic encephalomyelitis (EAE) by Perrin et al. ImmunolRes 14:189-99 (1995). EAE is an autoimmune disorder, induced by Th1cells directed against myelin antigens that provides an in vivo modelfor studying the role of B7-mediated costimulation in the induction of apathological immune response. Using a soluble fusion protein ligand forthe B7 receptors, as well as monoclonal antibodies specific for eitherCD80 or CD86, Perrin et al. demonstrated that B7 costimulation plays aprominent role in determining clinical disease outcome in EAE.

The interaction between B7 and CD28 is one of several co-stimulatorysignaling pathways that appear to be sufficient to trigger thematuration and proliferation of antigen specific T-cells. Lack ofco-stimulation, and the concomitant inadequacy of IL-2 production,prevent subsequent proliferation of the T cell and induce a state ofnon-reactivity termed “anergy”. A variety of viruses and tumors mayblock T cell activation and proliferation, leading to insufficientactivity or non-reactivity of the host's immune system to the infectedor transformed cells. Among a number of possible T-cell disturbances,anergy may be at least partly responsible for the failure of the host toclear the pathogenic or tumorgenic cells.

The use of the B7 protein to mediate anti-tumor immunity has beendescribed in Chen et al. Cell 71:1093-1102 (1992) and Townsend andAllison Science 259:368 (1993). Schwartz Cell 71:1065 (1992) reviews therole of CD28, CTLA-4, and B7 in IL-2 production and immunotherapy.Harding et al. Nature 356:607-609 (1994) demonstrates that CD28 mediatedsignaling co-stimulates murine T cells and prevents the induction ofanergy in T cell clones. See also U.S. Pat. Nos. 5,434,131, 5,770,197,and 5,773,253, and International Patent Application Nos. WO 93/00431, WO95/01994, WO 95/03408, WO 95/24217, and WO 95/33770.

From the foregoing, it was clear that T-cells required two types ofsignals from the antigen presenting cell (APC) for activation andsubsequent differentiation to effector function. First, there is anantigen specific signal generated by interactions between the TCR on theT-cell and MHC molecules presenting peptides on the APC. Second, thereis an antigen-independent signal that is mediated by the interaction ofCD28 with members of the B7 family (B7-1 (CD80) or B7-2 (CD86)). Exactlywhere CTLA-4 fit into the milieu of immune responsiveness was initiallyevasive. Murine CTLA-4 was first identified and cloned by Brunet et al.Nature 328:267-270 (1987), as part of a quest for molecules that arepreferentially expressed on cytotoxic T lymphocytes. Human CTLA-4 wasidentified and cloned shortly thereafter by Dariavach et al. Eur. J.Immunol. 18:1901-1905 (1988). The murine and human CTLA-4 moleculespossess approximately 76% overall sequence homology and approachcomplete sequence identity in their cytoplasmic domains (Dariavach etal. Eur. J. Immunol. 18:1901-1905 (1988)). CTLA-4 is a member of theimmunoglobulin (Ig) superfamily of proteins. The Ig superfamily is agroup of proteins that share key structural features of either avariable (V) or constant (C) domain of Ig molecules. Members of the Igsuperfamily include, but are not limited to, the immunoglobulinsthemselves, major histocompatibility complex (MHC) class molecules(i.e., MHC class I and II), and TCR molecules.

In 1991, Linsley et al. J. Exp. Med. 174:561-569 (1991), proposed thatCTLA-4 was a second receptor for B7. Similarly, Harper et al. J Immunol147:1037-44 (1991) demonstrated that the CTLA-4 and CD28 molecules areclosely related in both mouse and human as to sequence, messageexpression, gene structure, and chromosomal location. See also Balzanoet al. Int J Cancer Suppl 7:28-32 (1992). Further evidence of this rolearose through functional studies. For example, Lenschow et al. Science257:789-792 (1992) demonstrated that CTLA-4-Ig induced long termsurvival of pancreatic islet grafts. Freeman et al. Science 262:907-909(1993) examined the role of CTLA-4 in B7 deficient mice. Examination ofthe ligands for CTLA-4 are described in Lenschow et al. P.N.A.S.90:11054-11058 (1993). Linsley et al. Science 257:792-795 (1992)describes immunosuppression in vivo by a soluble form of CTLA-4. Linsleyet al. J Exp Med 176:1595-604 (1992) prepared antibodies that boundCTLA-4 and that were not cross-reactive with CD28 and concluded thatCTLA-4 is coexpressed with CD28 on activated T lymphocytes andcooperatively regulates T cell adhesion and activation by B7. Kuchroo etal. Cell 80:707-18 (1995) demonstrated that the B7-1 and B7-2costimulatory molecules differentially activated the Th1/Th2developmental pathways. Yi-qun et al. Int Immunol 8:37-44 (1996)demonstrated that there are differential requirements for co-stimulatorysignals from B7 family members by resting versus recently activatedmemory T cells towards soluble recall antigens. See also de Boer et al.Eur J Immunol 23:3120-5 (1993).

Several groups proposed alternative or distinct receptor/ligandinteractions for CTLA-4 as compared to CD28 and even proposed a thirdB-7 complex that was recognized by a BB1 antibody. See, for example,Hathcock et al. Science 262:905-7 (1993), Freeman et al. Science262:907-9 (1993), Freeman et al. J Exp Med 178:2185-92 (1993), Lenschowet al. Proc Natl Acad Sci USA 90:11054-8 (1993), Razi-Wolf et al. ProcNatl Acad Sci USA 90:11182-6 (1993), and Boussiotis et al. Proc NatlAcad Sci USA 90:11059-63 (1993). But, see, Freeman et al. J Immunol161:2708-15 (1998) who discuss finding that BB1 antibody binds amolecule that is identical to the cell surface form of CD74 and,therefore, the BB1 mAb binds to a protein distinct from B7-1, and thisepitope is also present on the B7-1 protein. Thus, this observationrequired the field to reconsider studies using BB1 mAb in the analysisof CD80 expression and function.

Beginning in 1993 and culminating in 1995, investigators began tofurther delineate the role of CTLA-4 in T-cell stimulation. First,through the use of monoclonal antibodies against CTLA-4, Walunas et al.Immunity 1:405-13 (1994) provided evidence that CTLA-4 can function as anegative regulator of T cell activation. Thereafter, Waterhouse et al.Science 270:985-988 (1995) demonstrated that mice deficient for CTLA-4accumulated T cell blasts with up-regulated activation markers in theirlymph nodes and spleens. The blast cells also infiltrated liver, heart,lung, and pancreas tissue, and amounts of serum immunoglobulin wereelevated and their T cells proliferated spontaneously and strongly whenstimulated through the T cell receptor, however, they were sensitive tocell death induced by cross-linking of the Fas receptor and by gammairradiation. Waterhouse et al. concluded that CTLA-4 acts as a negativeregulator of T cell activation and is vital for the control oflymphocyte homeostasis. In a comment in the same issue, Allison andKrummel Science 270:932-933 (1995), discussed the work of Waterhouse etal. as demonstrative that CTLA-4 acts to down regulate T-cellresponsiveness or has an inhibitory signaling role in T-cell activationand development. Tivol et al. Immunity 3:541-7 (1995) also generatedCTLA-4-deficient mice and demonstrated that such mice rapidly developlymphoproliferative disease with multiorgan lymphocytic infiltration andtissue destruction, with particularly severe myocarditis andpancreatitis. They concluded that CTLA-4 plays a key role indown-regulating T cell activation and maintaining immunologichomeostasis. Also, Krummel and Allison J Exp Med 182:459-65 (1995)further clarified that CD28 and CTLA-4 have opposing effects on theresponse of T cells to stimulation. They generated an antibody to CTLA-4and investigated the effects of its binding to CTLA-4 in a system usinghighly purified T cells. In their report, they showed that the presenceof low levels of B7-2 on freshly explanted T cells can partially inhibitT cell proliferation, and this inhibition was mediated by interactionswith CTLA-4. Cross-linking of CTLA-4 together with the TCR and CD28strongly inhibits proliferation and IL-2 secretion by T cells. Finally,the results showed that CD28 and CTLA-4 deliver opposing signals thatappear to be integrated by the T cell in determining the response toantigen. Thus, they concluded that the outcome of T cell antigenreceptor stimulation is regulated by CD28 costimulatory signals, as wellas inhibitory signals derived from CTLA-4. See also Krummel et al. IntImmunol 8:519-23 (1996) and U.S. Pat. No. 5,811,097 and InternationalPatent Application No. WO 97/20574.

A variety of additional experiments have been conducted furtherelucidating the above function of CTLA-4. For example, Walunas et al. JExp Med 183:2541-50 (1996), through the use of anti-CTLA-4 antibodies,suggested that CTLA-4 signaling does not regulate cell survival orresponsiveness to IL-2, but does inhibit CD28-dependent IL-2 production.Also, Perrin et al. J Immunol 157:1333-6 (1996), demonstrated thatanti-CTLA-4 antibodies in experimental allergic encephalomyelitis (EAE),exacerbated the disease and enhanced mortality. Disease exacerbation wasassociated with enhanced production of the encephalitogenic cytokinesTNF-alpha, IFN-gamma and IL-2. Thus, they concluded that CTLA-4regulates the intensity of the autoimmune response in EAE, attenuatinginflammatory cytokine production and clinical disease manifestations.See also Hurwitz et al. J Neuroimmunol 73:57-62 (1997) and Cepero et al.J Exp Med 188:199-204 (1998) (an anti-CTLA-4 hairpin ribozyme thatspecifically abrogates CTLA-4 expression after gene transfer into amurine T-cell model).

In addition, Blair et al. J Immunol 160:12-5 (1998) assessed thefunctional effects of a panel of CTLA-4 monoclonal antibodies (mAbs) onresting human CD4+ T cells. Their results demonstrated that some CTLA-4mAbs could inhibit proliferative responses of resting CD4+ cells andcell cycle transition from G0 to G1. The inhibitory effects of CTLA-4were evident within 4 h, at a time when cell surface CTLA-4 expressionremained undetectable. Other CTLA-4 mAbs, however, had no detectableinhibitory effects, indicating that binding of mAbs to CTLA-4 alone wasnot sufficient to mediate down-regulation of T cell responses.Interestingly, while IL-2 production was shut off, inhibitoryanti-CTLA-4 mAbs permitted induction and expression of the cell survivalgene bc1-X(L). Consistent with this observation, cells remained viableand apoptosis was not detected after CTLA-4 ligation.

In connection with anergy, Perez et al. Immunity 6:411-7 (1997)demonstrated that the induction of T cell anergy was prevented byblocking CTLA-4 and concluded that the outcome of antigen recognition byT cells is determined by the interaction of CD28 or CTLA-4 on the Tcells with B7 molecules. Also, Van Parijs et al. J Exp Med 186:1119-28(1997) examined the role of interleukin 12 and costimulators in T cellanergy in vivo and found that through inhibiting CTLA-4 engagementduring anergy induction, T cell proliferation was blocked, and full Th1differentiation was not promoted. However, T cells exposed totolerogenic antigen in the presence of both IL-12 and anti-CTLA-4antibody were not anergized, and behaved identically to T cells whichhave encountered immunogenic antigen. These results suggested that twoprocesses contribute to the induction of anergy in vivo: CTLA-4engagement, which leads to a block in the ability of T cells toproliferate, and the absence of a prototypic inflammatory cytokine,IL-12, which prevents the differentiation of T cells into Th1 effectorcells. The combination of IL-12 and anti-CTLA-4 antibody was sufficientto convert a normally tolerogenic stimulus to an immunogenic one.

In connection with infections, McCoy et al. J Exp Med 186:183-7 (1997)demonstrated that anti-CTLA-4 antibodies greatly enhanced andaccelerated the T cell immune response to Nippostrongylus brasiliensis,resulting in a profound reduction in adult worm numbers and earlytermination of parasite egg production. See also Murphy et al. J.Immunol. 161:4153-4160 (1998) (Leishmania donovani).

In connection with cancer, Kwon et al. PNAS USA 94:8099-103 (1997)established a syngeneic murine prostate cancer model and examined twodistinct manipulations intended to elicit an antiprostate cancerresponse through enhanced T cell costimulation: (i) provision of directcostimulation by prostate cancer cells transduced to express the B7.1ligand and (ii) in vivo antibody-mediated blockade of T cell CTLA-4,which prevents T cell down-regulation. It was demonstrated that in vivoantibody-mediated blockade of CTLA-4 enhanced antiprostate cancer immuneresponses. Also, Yang et al. Cancer Res 57:4036-41 (1997) investigatedwhether the blockade of the CTLA-4 function leads to enhancement ofantitumor T cell responses at various stages of tumor growth. Based onin vitro and in vivo results they found that CTLA-4 blockade intumor-bearing individuals enhanced the capacity to generate antitumorT-cell responses, but the expression of such an enhancing effect wasrestricted to early stages of tumor growth in their model. Further,Hurwitz et al. Proc Natl Acad Sci US A 95:10067-71 (1998) investigatedthe generation of a T cell-mediated antitumor response depends on T cellreceptor engagement by major histocompatibility complex/antigen as wellas CD28 ligation by B7. Certain tumors, such as the SM1 mammarycarcinoma, were refractory to anti-CTLA-4 immunotherapy. Thus, throughuse of a combination of CTLA-4 blockade and a vaccine consisting ofgranulocyte-macrophage colony-stimulating factor-expressing SM1 cells,regression of parental SM1 tumors was observed, despite theineffectiveness of either treatment alone. This combination therapyresulted in long-lasting immunity to SM1 and depended on both CD4(+) andCD8(+) T cells. The findings suggested that CTLA-4 blockade acts at thelevel of a host-derived antigen-presenting cell.

In connection with diabetes, Luhder et al. J Exp Med 187:427-32 (1998)injected an anti-CTLA-4 mAb into a TCR transgenic mouse model ofdiabetes at different stages of disease. They found that engagement ofCTLA-4 at the time when potentially diabetogenic T cells are firstactivated is a pivotal event; if engagement is permitted, invasion ofthe islets occurs, but remains quite innocuous for months. If not,insulitis is much more aggressive, and diabetes quickly ensues.

In connection with vaccine immunization, Horspool et al. J Immunol160:2706-14 (1998) found that intact anti-CTLA-4 mAb but not Fabfragments suppressed the primary humoral response to pCIA/beta galwithout affecting recall responses, indicating CTLA-4 activationinhibited Ab production but not T cell priming. Blockade of the ligandsfor CD28 and CTLA-4, CD80 (B7-1) and CD86 (B7-2), revealed distinct andnonoverlapping function. Blockade of CD80 at initial immunizationcompletely abrogated primary and secondary Ab responses, whereasblockade of CD86 suppressed primary but not secondary responses.Simultaneous blockade of CD80+CD86 was less effective at suppressing Abresponses than either alone. Enhancement of costimulation viacoinjection of B7-expressing plasmids augmented CTL responses but not Abresponses, and without evidence of Th1 to Th2 skewing. These findingssuggest complex and distinct roles for CD28, CTLA-4, CD80, and CD86 in Tcell costimulation following nucleic acid vaccination.

In connection with allograft rejection, Markees et al. J Clin Invest101:2446-55 (1998) found in a mouse model of skin allograft rejectionthat acceptance initially depended on the presence of IFN-gamma, CTLA-4,and CD4(+) T cells. Addition of anti-CTLA-4 or anti-IFN-gamma mAb to theprotocol was associated with prompt graft rejection, whereas anti-IL-4mAb had no effect.

In connection with the role of CTLA-4 in relation to CD28, Fallarino etal. J Exp Med 188:205-10 (1998) generated TCR transgenic/recombinaseactivating gene 2-deficient/CD28-wild-type or CD28-deficient mice whichwere immunized with an antigen-expressing tumor. Primed T cells fromboth types of mice produced cytokines and proliferated in response tostimulator cells lacking B7 expression. However, whereas the response ofCD28+/+ T cells was augmented by costimulation with B7-1, the responseof the CD28−/− T cells was strongly inhibited. This inhibition wasreversed by monoclonal antibody against B7-1 or CTLA-4. Thus, CTLA-4 canpotently inhibit T cell activation in the absence of CD28, indicatingthat antagonism of a TCR-mediated signal is sufficient to explain theinhibitory effect of CTLA-4. Also, Lin et al. J Exp Med 188:199-204(1998) studied rejection of heart allografts in CD28-deficient mice.H-2(q) hearts were transplanted into allogeneic wild-type orCD28-deficient mice (H-2(b)). Graft rejection was delayed inCD28-deficient compared with wild-type mice. Treatment of wild-typerecipients with CTLA-4-immunoglobulin (Ig), or with anti-B7-1 plusanti-B7-2 mAbs significantly prolonged allograft survival. In contrast,treatment of CD28-deficient mice with CTLA-4-Ig, anti-B7-1 plusanti-B7-2 mAbs, or a blocking anti-CTLA-4 mAb induced acceleration ofallograft rejection. This increased rate of graft rejection wasassociated with more severe mononuclear cell infiltration and enhancedlevels of IFN-gamma and IL-6 transcripts in donor hearts of untreatedwild-type and CTLA-4-Ig- or anti-CTLA-4 mAb-treated CD28-deficient mice.Thus, the negative regulatory role of CTLA-4 extends beyond itspotential ability to prevent CD28 activation through ligand competition.Even in the absence of CD28, CTLA-4 plays an inhibitory role in theregulation of allograft rejection.

Also, further characterization of the expression of CTLA-4 has beeninvestigated. For example, Alegre et al. J Immunol 157:4762-70 (1996)proposed that surface CTLA-4 is rapidly internalized, which may explainthe low levels of expression generally detected on the cell surface.They concluded that both CD28 and IL-2 play important roles in theup-regulation of CTLA-4 expression. In addition, the cell surfaceaccumulation of CTLA-4 appeared to be primarily regulated by its rapidendocytosis. Also, Castan et al. Immunology 90:265-71 (1997) based on insitu immunohistological analyses of the expression of CTLA-4, suggestedthat germinal center T cells, which were CTLA-4 positive, could beimportant to immune regulation.

Accordingly, in view of the broad and pivotal role that CTLA-4 appearsto possess in immune responsiveness, it would be desirable to generateantibodies to CTLA-4 that can be utilized effectively in immunotherapy.Moreover, it would be desirable to generate antibodies against CTLA-4that can be utilized in chronic diseases in which repeat administrationsof the antibodies are required.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided an antibody that is capable of binding CTLA-4, comprising aheavy chain variable region amino acid sequence that comprises acontiguous amino acid sequence from within an FR1 sequence through anFR3 sequence that is encoded by a human V_(H)3-33 family gene and thatcomprises at least one of the amino acid substitutions in the CDR1sequences, CDR2 sequences, or framework sequences shown in FIG. 2. In apreferred embodiment, the amino acid sequence comprises a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:63,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70. In anotherpreferred embodiment, the antibody further comprises a light chainvariable region amino acid sequence comprising a sequence selected fromthe group consisting of a sequence comprising SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQID NO:71.

In accordance with a second aspect of the present invention, there isprovided an antibody comprising a heavy chain amino acid sequencecomprising SEQ ID NO:1 and a light chain variable amino acid sequencecomprising SEQ ID NO:14.

In accordance with a third aspect of the present invention, there isprovided an antibody comprising a heavy chain amino acid sequencecomprising SEQ ID NO:2 and a light chain variable amino acid sequencecomprising SEQ ID NO:15.

In accordance with a fourth aspect of the present invention, there isprovided an antibody comprising a heavy chain amino acid sequencecomprising SEQ ID NO:4 and a light chain variable amino acid sequencecomprising SEQ ID NO:17.

In accordance with a fifth aspect of the present invention, there isprovided an isolated human monoclonal antibody that is capable ofbinding to CTLA-4. In a preferred embodiment, antibody is capable ofcompeting for binding with CTLA-4 with an antibody selected from thegroup consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1,11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In another preferredembodiment, the antibody possesses a substantially similar bindingspecificity to CTLA-4 as an antibody selected from the group consistingof 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1,11.7.1, 12.3.1.1, and 12.9.1.1. In another preferred embodiment, theantibody is selected from the group consisting of 3.1.1, 4.1.1, 4.8.1,4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and12.9.1.1. In another preferred embodiment, the antibody is not crossreactive with CTLA-4 from lower mammalian species, preferably the lowermammalian species comprises mouse, rat, and rabbit and more preferablymouse and rat. In another preferred embodiment, the antibody is crossreactive with CTLA-4 from primates, preferably the primates comprisecynomolgous and rhesus monkeys. In another preferred embodiment, theantibody possesses a selectivity for CTLA-4 over CD28, B7-2, CD44, andhIgG1 of greater than about 100:1 and preferably about 500:1 or greater.In another preferred embodiment, the binding affinity of the antibody isabout 10⁻⁹ M or greater and preferably about 10⁻¹⁰ M or greater. Inanother preferred embodiment, the antibody inhibits binding betweenCTLA-4 and B7-2 with an IC₅₀ of lower than about 100 nM and preferablylower than about 0.38 nM. In another preferred embodiment, the antibodyinhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower thanabout 100 nM or greater and preferably lower than about 0.50 nM. Inanother preferred embodiment, the antibody enhances IL-2 production in aT cell blast/Raji assay by about 500 pg/ml or greater and preferably byabout 3846 pg/ml or greater. In another preferred embodiment, theantibody enhances IFN-γ production in a T cell blast/Raji assay by about500 pg/ml or greater and preferably by about 1233 pg/ml or greater. Inanother preferred embodiment, the antibody enhances IL-2 production in ahPBMC or whole blood superantigen assay by about 500 pg/ml or greater.In another preferred embodiment, the antibody enhances IL-2 productionin a hPBMC or whole blood superantigen assay by about 500 pg/ml orpreferably 1500 pg/ml or greater or by greater than about 30% orpreferably 50% relative to control.

In accordance with a sixth aspect of the present invention, there isprovided a humanized antibody that possesses a substantially similarbinding specificity to CTLA-4 as an antibody selected from the groupconsisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1,11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In a preferredembodiment, the antibody is not cross reactive with CTLA-4 from lowermammalian species, preferably the lower mammalian species comprisesmouse, rat, and rabbit and preferably mouse and rat. In anotherpreferred embodiment, the antibody is cross reactive with CTLA-4 fromprimates, preferably the primates comprise cynomolgous and rhesusmonkeys. In another preferred embodiment, the antibody possesses aselectivity for CTLA-4 over CD28, B7-2, CD44, and hIgG1 of greater thanabout 100:1 and preferably about 500:1 or greater. In another preferredembodiment, the binding affinity of the antibody is about 10⁻⁹ M orgreater and preferably about 10⁻¹⁰ M or greater. In another preferredembodiment, the antibody inhibits binding between CTLA-4 and B7-2 withan IC₅₀ of lower than about 100 nM and preferably lower than about 0.38nM. In another preferred embodiment, the antibody inhibits bindingbetween CTLA-4 and B7-1 with an IC₅₀ of lower than about 100 nM orgreater and preferably lower than about 0.50 nM. In another preferredembodiment, the antibody enhances IL-2 production in a T cell blast/Rajiassay by about 500 pg/ml or greater and preferably by about 3846 pg/mlor greater. In another preferred embodiment, the antibody enhances IFN-γproduction in a T cell blast/Raji assay by about 500 pg/ml or greaterand preferably by about 1233 pg/ml or greater. In another preferredembodiment, the antibody induces IL-2 production in a hPBMC or wholeblood superantigen assay by about 500 pg/ml or greater. In anotherpreferred embodiment, the antibody enhances IL-2 production in a hPBMCor whole blood superantigen assay by about 500 pg/ml or preferably 1500pg/ml or greater or by greater than about 30% or preferably 50% relativeto control.

In accordance with a seventh aspect of the present invention, there isprovided an antibody that binds to CTLA-4, comprising a heavy chainamino acid sequence comprising human FR1, FR2, and FR3 sequences encodedby a human V_(H) 3-33 gene family operably linked in frame with a CDR1,a CDR2, and a CDR3 sequence, the CDR1, CDR2, and CDR3 sequences beingindependently selected from the CDR1, CDR2, and CDR3 sequencesillustrated in FIG. 2. In a preferred embodiment, the antibody of claim32, further comprising any of the somatic mutations to the FR1, FR2, andFR3 sequences as illustrated in FIG. 2.

In accordance with an eighth aspect of the present invention, there isprovided an antibody that binds to CTLA-4, comprising a heavy chainamino acid sequence comprising human FR1, FR2, and FR3 sequences encodedby a human V_(H) 3-33 gene family operably linked in frame with a CDR1,a CDR2, and a CDR3 sequence, which antibody has the followingproperties: a binding affinity for CTLA-4 of about 10⁻⁹ or greater;inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of about 100 nM orlower; inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of about100 nM or lower; and enhances cytokine production in an assay of human Tcells by 500 pg/ml or greater.

In accordance with a ninth aspect of the present invention, there isprovided an antibody that binds to CTLA-4, comprising a heavy chainamino acid sequence comprising FR1, FR2, and FR3 sequences operablylinked in frame with a CDR1, a CDR2, and a CDR3 sequence independentlyselected from the CDR1, CDR2, and CDR3 sequences illustrated in FIGS. 2and 3, which antibody has the following properties: a binding affinityfor CTLA-4 of about 10⁻⁹ or greater; inhibits binding between CTLA-4 andB7-1 with an IC₅₀ of about 100 nM or lower; inhibits binding betweenCTLA-4 and B7-2 with an 10₅₀ of about 100 nM or lower; and enhancescytokine production in an assay of human T cells by 500 pg/ml orgreater.

In accordance with a tenth aspect of the present invention, there isprovided a cell culture system for assaying T cell stimulation,comprising a culture of human T cell blasts co-cultured with a Raji cellline. In a preferred embodiment, the T cell blasts are washed prior toculture with the Raji cell line.

In accordance with an eleventh aspect of the present invention, there isprovided an assay for measuring T cell stimulation, comprising:providing a culture of human T cell blasts and a Raji cell line;contacting the culture with an agent; and measuring cytokine productionby the culture.

In accordance with an twelfth aspect of the present invention, there isprovided a functional assay for screening a moiety for T cellstimulatory function, comprising: providing a culture of human T cellblasts and a Raji cell line; contacting the culture with the moiety; andassessing cytokine production by the culture.

In accordance with a thirteenth aspect of the present invention, thereis provided a T cell stimulatory assay for CTLA-4 inhibitory function,comprising contacting a culture comprising human T cell blasts and aRaji cell line with an agent and assessing cytokine production by theculture.

In accordance with a fourteenth aspect of the present invention, thereis provided a method for screening an agent for T cell stimulatoryactivity, comprising: contacting the agent with a cell culturecomprising human T cell blasts and a Raji cell line; and assessingcytokine production by the culture.

In each of the tenth through the fourteenth aspects of the presentinvention, in a preferred embodiment, the T cell blasts are washed priorto culture with the Raji cell line. In another preferred embodiment, thecytokine is IL-2 or IFN-γ. In a preferred embodiment, cytokineproduction is measured in supernatant isolated from the culture. In apreferred embodiment, the agent is an antibody and preferably binds toCTLA-4.

In accordance with a fifteenth aspect of the present invention, there isprovided an assay for measuring T cell stimulation, comprising:providing a population of human peripheral blood mononuclear cells orhuman whole blood stimulated with staphylococcus enterotoxin A;contacting the culture with an agent; and measuring cytokine productionby the cell population.

In accordance with a sixteenth aspect of the present invention, there isprovided a functional assay for screening a moiety for T cellstimulatory function, comprising: providing a population of humanperipheral blood mononuclear cells or human whole blood stimulated withstaphylococcus enterotoxin A; contacting the culture with the moiety;and assessing cytokine production by the cell population.

In accordance with a seventeenth aspect of the present invention, thereis provided a T cell stimulatory assay for CTLA-4 inhibitory function,comprising contacting a population of human peripheral blood mononuclearcells or human whole blood stimulated with staphylococcus enterotoxin Awith an agent and assessing cytokine production by the cell population.

In accordance with an eighteenth aspect of the present invention, thereis provided a method for screening an agent for T cell stimulatoryactivity, comprising: contacting the agent with a population of humanperipheral blood mononuclear cells or human whole blood stimulated withstaphylococcus enterotoxin A; and assessing cytokine production by thecell population.

In each of the fifteenth through the eighteenth aspects of the presentinvention, in a preferred embodiment, the cytokine is IL-2. In anotherpreferred embodiment, cytokine production is measured in supernatantisolated from the culture. In a preferred embodiment, the agent is anantibody and preferably binds to CTLA-4.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIGS. 1A-1M provide a series of nucleotide and amino acid sequences ofheavy chain and kappa light chain immunoglobulin molecules in accordancewith the invention: 4.1.1 (FIG. 1A), 4.8.1 (FIG. 1B), 4.1-4.3 (FIG. 1C),6.1.1 (FIG. 1D), 3.1.1 (FIG. 1E), 4.1-0.2 (FIG. 1F), 2.1.3 (FIG. 1G),4.1-3.1 (FIG. 1H), 11.2.1 (FIG. 1I), 11.6.1 (FIG. 1J), 11.7.1 (FIG. 1K),12.3.1.1 (FIG. 1L), and 12.9.1.1 (FIG. 1M).

FIGS. 2A-D provide a sequence alignment between the predicted heavychain amino acid sequences from the clones 4.1.1 (SEQ ID NO: 74), 4.8.1(SEQ ID NO: 75), 4.1-4.3 (SEQ ID NO: 78), 6.1.1 (SEQ ID NO: 79), 3.1.1(SEQ ID NO: 73), 4.1-0.2 (SEQ ID NO: 76), 4.1-3.1 (SEQ ID NO: 77),11.2.1 (SEQ ID NO: 80), 11.6.1 (SEQ ID NO: 81), 11.7.1 (SEQ ID NO: 82),12.3.1.1 (SEQ ID NO: 83), and 12.9.1.1 (SEQ ID NO: 84) and the germlineDP-50 (3-33) amino acid sequence (SEQ ID NO: 72). Differences betweenthe DP-50 germline sequence and that of the sequence in the clones areindicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibodies as shaded.

FIG. 3 provides a sequence alignment between the predicted heavy chainamino acid sequence of the clone 2.1.3 (SEQ ID NO: 86) and the germlineDP-65 (4-31) amino acid sequence (SEQ ID NO: 85). Differences betweenthe DP-65 germline sequence and that of the sequence in the clone areindicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 4 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clones 4.1.1 (SEQ ID NO: 88), 4.8.1(SEQ ID NO: 89), 4.1-4.3 (SEQ ID NO: 90), 6.1.1 (SEQ ID NO: 91), 4.10.2(SEQ ID NO: 92), and 4.13.1 (SEQ ID NO: 93) and the germline A27 aminoacid sequence (SEQ ID NO: 87). Differences between the A27 germlinesequence and that of the sequence in the clone are indicated in bold.The Figure also shows the positions of the CDR1, CDR2, and CDR3sequences of the antibody as underlined. Apparent deletions in the CDR1sof clones 4.8.1, 4.14.3, and 6.1.1 are indicated with “0s”.

FIG. 5 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clones 3.1.1 (SEQ ID NO: 95), 11.2.1(SEQ ID NO: 96), 11.6.1 (SEQ ID NO: 97), and 11.7.1 (SEQ ID NO: 98) andthe germline 012 amino acid sequence (SEQ ID NO: 94). Differencesbetween the 012 germline sequence and that of the sequence in the cloneare indicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 6 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 2.1.3 (SEQ ID NO: 112) and thegermline A10/A26 amino acid sequence (SEQ ID NO: 99). Differencesbetween the A10/A26 germline sequence and that of the sequence in theclone are indicated in bold. The Figure also shows the positions of theCDR1, CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 7 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 12.3.1 (SEQ ID NO: 114) and thegermline A17 amino acid sequence (SEQ ID NO: 113). Differences betweenthe A17 germline sequence and that of the sequence in the clone areindicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 8 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 12.9.1 (SEQ ID NO: 116) and thegermline A3/A19 amino acid sequence (SEQ ID NO: 115). Differencesbetween the A3/A19 germline sequence and that of the sequence in theclone are indicated in bold. The Figure also shows the positions of theCDR1, CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 9 provides a summary of N-terminal amino acid sequences generatedthrough direct protein sequencing of the heavy and light chains of theantibodies.

FIGS. 10A-10D provide certain additional characterizing informationabout certain of the antibodies in accordance with the invention. InFIG. 10A, data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3,and 6.1.1 is summarized. Data related to concentration, isoelectricfocusing (IEF), SDS-PAGE, size exclusion chromatography, liquidchromatography/mass spectroscopy (LCMS), mass spectroscopy (MALDI), andlight chain N-terminal sequences is provided. Additional detailedinformation related to IEF is provided in FIG. 10B; related to SDS-PAGEis provided in 10C; and SEC of the 4.1.1 antibody in 10D.

FIGS. 11A and 11B show the expression of B7-1 (11A) and B7-2 (11B) onRaji cells using anti-CD80-PE and anti-CD86-PE mAbs.

FIG. 12 shows the concentration dependent enhancement of IL-2 productionin the T cell blast/Raji assay induced by anti-CTLA-4 blockingantibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1).

FIG. 13 shows the concentration dependent enhancement of IFN-γproduction in the T cell blast/Raji assay induced by anti-CTLA-4blocking antibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1)(same donor T cells).

FIG. 14 shows the mean enhancement of IL-2 production in T cells from 6donors induced by anti-CTLA-4 blocking antibodies in the T cellblast/Raji assay.

FIG. 15 shows the mean enhancement of IFN-γ production in T cells from 6donors induced by anti-CTLA-4 blocking antibodies in the T cellblast/Raji assay.

FIG. 16 shows the enhancement of IL-2 production in hPBMC from 5 donorsinduced by anti-CTLA-4 blocking mAbs as measured at 72 hours afterstimulation with SEA.

FIG. 17 shows the enhancement of IL-2 production in whole blood from 3donors induced by anti-CTLA-4 blocking mAbs as measured at 72 and 96hours after stimulation with SEA.

FIG. 18 shows the inhibition of tumor growth with an anti-murine CTLA-4antibody in a murine fibrosarcoma tumor model.

FIG. 19 shows enhancement of IL-2 production induced by anti-CTLA4antibodies (4.1.1 and 11.2.1) of the invention in a 72 hour T blast/Rajiand Superantigen (whole blood and peripheral blood mononuclear cellsfrom 6 donors) assays.

FIG. 20 shows dose dependent enhancement of IL-2 production induced byanti-CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 hour Tblast/Raji assay.

FIG. 21 shows dose dependent enhancement of IL-2 production induced byanti-CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 hourSuperantigen whole blood assay stimulated with 100 ng/ml superantigen.

FIGS. 22A-22S provide a series of additional nucleotide and amino acidsequences of the following anti-CTLA-4 antibody chains: full length4.1.1 heavy chain (cDNA 22(a), genomic 22(b), and amino acid 22(c)),full length aglycosylated 4.1.1 heavy chain (cDNA 22(d) and amino acid22(e)), 4.1.1 light chain (cDNA 22(f) and amino acid 22(g)), full length4.8.1 heavy chain (cDNA 22(h) and amino acid 22(i)), 4.8.1 light chain(cDNA 22(j) and amino acid 22(k)), full length 6.1.1 heavy chain (cDNA22(l) and amino acid 22(m)), 6.1.1 light chain (cDNA 22(n) and aminoacid 22(o)), full length 11.2.1 heavy chain (cDNA 22(p) and amino acid22(q)), and 11.2.1 light chain (cDNA 22 (r) and amino acid 22(s)).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there are provided fully humanmonoclonal antibodies against human CTLA-4. Nucleotide sequencesencoding and amino acid sequences comprising heavy and light chainimmunoglobulin molecules, particularly sequences corresponding to acontiguous heavy and light chain sequences from FR1 and CDR1 throughCDR3 and FR4, are provided. Further provided are antibodies havingsimilar binding properties and antibodies (or other antagonists) havingsimilar functionality as antibodies disclosed herein. Hybridomasexpressing such immunoglobulin molecules and monoclonal antibodies arealso provided.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)), which isincorporated herein by reference. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g. free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” as used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules represented in FIG. 1, as well asantibody molecules formed by combinations comprising the heavy chainimmunoglobulin molecules with light chain immunoglobulin molecules, suchas the kappa light chain immunoglobulin molecules, and vice versa, aswell as fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5′ end; the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. More preferred families are: serine and threonine arealiphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Assays are described in detailherein. Fragments or analogs of antibodies or immunoglobulin moleculescan be readily prepared by those of ordinary skill in the art. Preferredamino- and carboxy-termini of fragments or analogs occur near boundariesof functional domains. Structural and functional domains can beidentified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to CTLA-4, under suitablebinding conditions, (2) ability to block CTLA-4 binding with itsreceptors, or (3) ability to inhibit CTLA-4 expressing cell growth invitro or in vivo. Typically, polypeptide analogs comprise a conservativeamino acid substitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drus with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of:—CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the dissociation constant is ≦1 μM, preferably ≦100 nM and mostpreferably ≦10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new moleculardesign for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992)).Production of bispecific antibodies can be a relatively labor intensiveprocess compared with production of conventional antibodies and yieldsand degree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid certain of the problems associated withantibodies that possess murine or rat variable and/or constant regions.The presence of such murine or rat derived proteins can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, it has been postulatedthat one can develop humanized antibodies or generate fully humanantibodies through the introduction of human antibody function into arodent so that the rodent would produce antibodies having fully humansequences.

Human Antibodies

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs) an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derivatized Mabs and thus to increase the efficacy andsafety of the administered antibodies. The use of fully human antibodiescan be expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated antibodyadministrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human Mabs with thedesired specificity could be readily produced and selected.

This general strategy was demonstrated in connection with our generationof the first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineeredwith yeast artificial chromosomes (YACs) containing 245 kb and 190kb-sized germline configuration fragments of the human heavy chain locusand kappa light chain locus, respectively, which contained core variableand constant region sequences. Id. The human Ig containing YACs provedto be compatible with the mouse system for both rearrangement andexpression of antibodies and were capable of substituting for theinactivated mouse Ig genes. This was demonstrated by their ability toinduce B-cell development, to produce an adult-like human repertoire offully human antibodies, and to generate antigen-specific human Mabs.These results also suggested that introduction of larger portions of thehuman Ig loci containing greater numbers of V genes, additionalregulatory elements, and human Ig constant regions might recapitulatesubstantially the full repertoire that is characteristic of the humanhumoral response to infection and immunization. The work of Green et al.was recently extended to the introduction of greater than approximately80% of the human antibody repertoire through introduction of megabasesized, germline configuration YAC fragments of the human heavy chainloci and kappa light chain loci, respectively, to produce XenoMouse™mice. See Mendez et al. Nature Genetics 15:146-156 (1997), Green andJakobovits J. Exp. Med. 188:483-495 (1998), and U.S. patent applicationSer. No. 08/759,620, filed Dec. 3, 1996, the disclosures of which arehereby incorporated by reference.

Such approach is further discussed and delineated in U.S. patentapplication Ser. No. 07/466,008, filed Jan. 12, 1990, Ser. No.07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24,1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No.08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430, 938, Apr. 27, 1995, Ser. No.08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995,Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun.5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,and Ser. No. 08/759,620, filed Dec. 3, 1996. See also Mendez et al.Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998). See also European Patent No., EP 0 463 151 B1, grantpublished Jun. 12, 1996, International Patent Application No., WO94/02602, published Feb. 3, 1994, International Patent Application No.,WO 96/34096, published Oct. 31, 1996, and WO 98/24893, published Jun.11, 1998. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal This approach is describedin U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat. No.5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn,and GenPharm International U.S. patent application Ser. No. 07/574,748,filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No.07/810,279, filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18,1992, Ser. No. 07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860,filed Dec. 16, 1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No.08/096,762, filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18,1993, Ser. No. 08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699,filed Dec. 10, 1993, Ser. No. 08/209,741, filed Mar. 9, 1994, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Application Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

The inventors of Surani et al., cited above and assigned to the MedicalResearch Counsel (the “MRC”), produced a transgenic mouse possessing anIg locus through use of the minilocus approach. The inventors on theGenPharm International work, cited above, Lonberg and Kay, following thelead of the present inventors, proposed inactivation of the endogenousmouse Ig locus coupled with substantial duplication of the Surani et al.work.

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. Commensurately, however, a significantdisadvantage of the minilocus approach is that, in theory, insufficientdiversity is introduced through the inclusion of small numbers of V, D,and J genes. Indeed, the published work appears to support this concern.B-cell development and antibody production of animals produced throughuse of the minilocus approach appear stunted. Therefore, researchsurrounding the present invention has consistently been directed towardsthe introduction of large portions of the Ig locus in order to achievegreater diversity and in an effort to reconstitute the immune repertoireof the animals.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against CTLA-4 in order to vitiate concerns and/or effects ofHAMA or HACA response.

Humanization and Display Technologies

As was discussed above in connection with human antibody generation,there are advantages to producing antibodies with reducedimmunogenicity. To a degree, this can be accomplished in connection withtechniques of humanization and display techniques using appropriatelibraries. It will be appreciated that murine antibodies or antibodiesfrom other species can be humanized or primatized using techniques wellknown in the art. See e.g., Winter and Harris Immunol Today 14:43-46(1993) and Wright et al. Crit. Reviews in Immunol. 12125-168 (1992). Theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190 and U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). Also, the use of Ig cDNA for construction of chimericimmunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439(1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions to genes may be found in Kabat et al. (1991) Sequencesof Proteins of Immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3 and IgG4.Particularly preferred isotypes for antibodies of the invention are IgG2and IgG4. Either of the human light chain constant regions, kappa orlambda, may be used. The chimeric, humanized antibody is then expressedby conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

In one approach, consensus sequences encoding the heavy and light chainJ regions may be used to design oligonucleotides for use as primers tointroduce useful restriction sites into the J region for subsequentlinkage of V region segments to human C region segments. C region cDNAcan be modified by site directed mutagenesis to place a restriction siteat the analogous position in the human sequence.

Expression vectors include plasmids, retroviruses, cosmids, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)); native Igpromoters, etc.

Further, human antibodies or antibodies from other species can begenerated through display-type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau PNAS USA 94:4937-4942(1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988)(phage display), Scott TIBS 17:241-245 (1992), Cwirla et al. PNAS USA87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085(1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell andMcCafferty TIBTECH 10:80-84 (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated to CTLA-4 expressingcells, CTLA-4 itself, forms of CTLA-4, epitopes or peptides thereof, andexpression libraries thereto (see e.g. U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed above.

Additional Criteria for Antibody Therapeutics

As will be appreciated, it is generally not desirable to kill CTLA-4expressing cells. Rather, one generally desires to simply inhibit CTLA-4binding with its ligands to mitigate T cell down regulation. One of themajor mechanisms through which antibodies kill cells is through fixationof complement and participation in CDC. The constant region of anantibody plays an important role in connection with an antibody'sability to fix complement and participate in CDC. Thus, generally oneselects the isotype of an antibody to either provide the ability ofcomplement fixation, or not. In the case of the present invention,generally, as mentioned above, it is generally not preferred to utilizean antibody that kills the cells. There are a number of isotypes ofantibodies that are capable of complement fixation and CDC, including,without limitation, the following: murine IgM, murine IgG2a, murineIgG2b, murine IgG3, human IgM, human IgG1, and human IgG3. Thoseisotypes that do not include, without limitation, human IgG2 and humanIgG4.

It will be appreciated that antibodies that are generated need notinitially possess a particular desired isotype but, rather, the antibodyas generated can possess any isotype and the antibody can be isotypeswitched thereafter using conventional techniques that are well known inthe art. Such techniques include the use of direct recombinanttechniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusiontechniques (see e.g., U.S. patent application Ser. No. 08/730,639, filedOct. 11, 1996), among others.

In the cell-cell fusion technique, a myeloma or other cell line isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

By way of example, the majority of the CTLA-4 antibodies discussedherein are human anti-CTLA-4 IgG2 antibody. Since such antibodiespossess desired binding to the CTLA-4 molecule, any one of suchantibodies can be readily isotype switched to generate a human IgG4isotype, for example, while still possessing the same variable region(which defines the antibody's specificity and some of its affinity).

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain additional “functional” attributes thatare desired through isotype switching.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto CTLA-4, the design of other therapeutic modalities including otherantibodies, other antagonists, or chemical moieties other thanantibodies is facilitated. Such modalities include, without limitation,antibodies having similar binding activity or functionality, advancedantibody therapeutics, such as bispecific antibodies, immunotoxins, andradiolabeled therapeutics, generation of peptide therapeutics, genetherapies, particularly intrabodies, antisense therapeutics, and smallmolecules. Furthermore, as discussed above, the effector function of theantibodies of the invention may be changed by isotype switching to anIgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeuticuses.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecifics, immunotoxins, or radiolabels, for example.

In connection with bispecific antibodies, bispecific antibodies can begenerated that comprise (i) two antibodies one with a specificity toCTLA-4 and another to a second molecule that are conjugated together,(ii) a single antibody that has one chain specific to CTLA-4 and asecond chain specific to a second molecule, or (iii) a single chainantibody that has specificity to CTLA-4 and the other molecule. Suchbispecific antibodies can be generated using techniques that are wellknown for example, in connection with (i) and (ii) see e.g., Fanger etal. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra. and inconnection with (iii) see e.g., Traunecker et al. Int. J. Cancer(Suppl.) 7:51-52 (1992).

In addition, “Kappabodies” (Ill et al. “Design and construction of ahybrid immunoglobulin domain with properties of both heavy and lightchain variable regions” Protein Eng 10:949-57 (1997)), “Minibodies”(Martin et al. “The affinity-selection of a minibody polypeptideinhibitor of human interleukin-6” EMBO J. 13:5303-9 (1994)), “Diabodies”(Holliger et al. “‘Diabodies’: small bivalent and bispecific antibodyfragments” PNAS USA 90:6444-6448 (1993)), or “Janusins” (Traunecker etal. “Bispecific single chain molecules (Janusins) target cytotoxiclymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) andTraunecker et al. “Janusin: new molecular design for bispecificreagents” Int J Cancer Suppl 7:51-52 (1992)) may also be prepared.

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing CTLA-4, and particularly those cells inwhich the antibodies of the invention are effective.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to CTLA-4 and antibodiesthereto, such as the antibodies of the invention (as discussed below inconnection with small molecules) or screening of peptide libraries,therapeutic peptides can be generated that are directed against CTLA-4.Design and screening of peptide therapeutics is discussed in connectionwith Houghten et al. Biotechniques 13:412-421 (1992), Houghten PNAS USA82:5131-5135 (1985), Pinalla et al. Biotechniques 13:901-905 (1992),Blake and Litzi-Davis BioConjugate Chem. 3:510-513 (1992) Immunotoxinsand radiolabeled molecules can also be prepared, and in a similarmanner, in connection with peptidic moieties as discussed above inconnection with antibodies.

Important information related to the binding of an antibody to anantigen can be gleaned through phage display experimentation. Suchexperiments are generally accomplished through panning a phage libraryexpressing random peptides for binding with the antibodies of theinvention to determine if peptides can be isolated that bind. Ifsuccessful, certain epitope information can be gleaned from the peptidesthat bind.

In general, phage libraries expressing random peptides can be purchasedfrom New England Biolabs (7-mer and 12-mer libraries, Ph.D.-7 Peptide7-mer Library Kit and Ph.D.-12 Peptide 12-mer Library Kit, respectively)based on a bacteriophage M13 system. The 7-mer library represents adiversity of approximately 2.0×10⁹ independent clones, which representsmost, if not all, of the 20⁷=1.28×10⁹ possible 7-mer sequences. The12-mer library contains approximately 1.9×10⁹ independent clones andrepresents only a very small sampling of the potential sequence space of20¹²=4.1×10¹⁵ 12-mer sequences. Each of 7-mer and 12-mer libraries arepanned or screened in accordance with the manufacturer's recommendationsin which plates were coated with an antibody to capture the appropriateantibody (a goat anti-human IgG Fc for an IgG antibody for example)followed by washing. Bound phage are eluted with 0.2 M glycine-HCl, pH2.2. After 3 rounds of selection/amplification at constant stringency(0.5% Tween), through use of DNA sequencing, one can characterize clonesfrom the libraries that are reactive with one or more of the antibodies.Reactivity of the peptides can be determined by ELISA. For an additionaldiscussion of epitope analysis of peptides see also Scott, J. K. andSmith, G. P. Science 249:386-390 (1990); Cwirla et al. PNAS USA87:6378-6382 (1990); Felici et al. J. Mol. Biol. 222:301-310 (1991), andKuwabara et al. Nature Biotechnology 15:74-78 (1997).

The design of gene and/or antisense therapeutics through conventionaltechniques is also facilitated through the present invention. Suchmodalities can be utilized for modulating the function of CTLA-4. Inconnection therewith the antibodies of the present invention facilitatedesign and use of functional assays related thereto. A design andstrategy for antisense therapeutics is discussed in detail inInternational Patent Application No. WO 94/29444. Design and strategiesfor gene therapy are well known. However, in particular, the use of genetherapeutic techniques involving intrabodies could prove to beparticularly advantageous. See e.g., Chen et al. Human Gene Therapy5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997). General designof and considerations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137. Genetic materialsencoding an antibody of the invention (such as the 4.1.1, 4.8.1, or6.1.1, or others) may be included in a suitable expression system(whether viral, attenuated viral, non-viral, naked, or otherwise) andadministered to a host for in vivo generation of the antibody in thehost.

Small molecule therapeutics can also be envisioned in accordance withthe present invention. Drugs can be designed to modulate the activity ofCTLA-4 based upon the present invention. Knowledge gleaned from thestructure of the CTLA-4 molecule and its interactions with othermolecules in accordance with the present invention, such as theantibodies of the invention, CD28, B7, B7-1, B7-2, and others can beutilized to rationally design additional therapeutic modalities. In thisregard, rational drug design techniques such as X-ray crystallography,computer-aided (or assisted) molecular modeling (CAMM), quantitative orqualitative structure-activity relationship (QSAR), and similartechnologies can be utilized to focus drug discovery efforts. Rationaldesign allows prediction of protein or synthetic structures which caninteract with the molecule or specific forms thereof which can be usedto modify or modulate the activity of CTLA-4. Such structures can besynthesized chemically or expressed in biological systems. This approachhas been reviewed in Capsey et al. Genetically Engineered HumanTherapeutic Drugs (Stockton Press, NY (1988)). Indeed, the rationaldesign of molecules (either peptides, peptidomimetics, small molecules,or the like) based upon known, or delineated, structure-activityrelationships with other molecules (such as antibodies in accordancewith the invention) has become generally routine. See, e.g., Fry et al.“Specific, irreversible inactivation of the epidermal growth factorreceptor and erbB2, by a new class of tyrosine kinase inhibitor” ProcNatl Acad Sci US A 95:12022-7 (1998); Hoffman et al. “A model of Cdc25phosphatase catalytic domain and Cdk-interaction surface based on thepresence of a rhodanese homology domain” J Mol Biol 282:195-208 (1998);Ginalski et al. “Modelling of active forms of protein kinases: p38—acase study” Acta Biochim Pol 44:557-64 (1997); Jouko et al.“Identification of csk tyrosine phosphorylation sites and a tyrosineresidue important for kinase domain structure” Biochem J 322:927-35(1997); Singh et al. “Structure-based design of a potent, selective, andirreversible inhibitor of the catalytic domain of the erbB receptorsubfamily of protein tyrosine kinases” J Med Chem 40:1130-5 (1997);Mandel et al. “ABGEN: a knowledge-based automated approach for antibodystructure modeling” Nat Biotechnol 14:323-8 (1996); Monfardini et al.“Rational design, analysis, and potential utility of GM-CSF antagonists”Proc Assoc Am Physicians 108:420-31 (1996); Furet et al. “Modellingstudy of protein kinase inhibitors: binding mode of staurosporine andorigin of the selectivity of CGP 52411” J Comput Aided Mol Des 9:465-72(1995).

Further, combinatorial libraries can be designed and sythesized and usedin screening programs, such as high throughput screening efforts.

Therapeutic Administration and Formulations

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15^(th) ed, Mack Publishing Company, Easton,Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related to excipientsand carriers well known to pharmaceutical chemists.

Preparation of Antibodies

Antibodies in accordance with the invention are preferably preparedthrough the utilization of a transgenic mouse that has a substantialportion of the human antibody producing genome inserted but that isrendered deficient in the production of endogenous, murine, antibodies.Such mice, then, are capable of producing human immunoglobulin moleculesand antibodies and are deficient in the production of murineimmunoglobulin molecules and antibodies. Technologies utilized forachieving the same are disclosed in the patents, applications, andreferences disclosed in the Background, herein. In particular, however,a preferred embodiment of transgenic production of mice and antibodiestherefrom is disclosed in U.S. patent application Ser. No. 08/759,620,filed Dec. 3, 1996, the disclosure of which is hereby incorporated byreference. See also Mendez et al. Nature Genetics 15:146-156 (1997), thedisclosure of which is hereby incorporated by reference.

Through use of such technology, we have produced fully human monoclonalantibodies to a variety of antigens. Essentially, we immunize XenoMouse™lines of mice with an antigen of interest, recover lymphatic cells (suchas B-cells) from the mice that express antibodies, fuse such recoveredcells with a myeloid-type cell line to prepare immortal hybridoma celllines, and such hybridoma cell lines are screened and selected toidentify hybridoma cell lines that produce antibodies specific to theantigen of interest. We utilized these techniques in accordance with thepresent invention for the preparation of antibodies specific to CTLA-4.Herein, we describe the production of multiple hybridoma cell lines thatproduce antibodies specific to CTLA-4. Further, we provide acharacterization of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

The antibodies derived from hybridoma cell lines discussed herein aredesignated 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1,11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. Each of the antibodies producedby the aforementioned cell lines are either fully human IgG2 or IgG4heavy chains with human kappa light chains. In general, antibodies inaccordance with the invention possess very high affinities, typicallypossessing Kd's of from about 10⁻⁹ through about 10⁻¹¹ M, when measuredby either solid phase or solution phase.

As will be appreciated, antibodies in accordance with the presentinvention can be expressed in cell lines other than hybridoma celllines. Sequences encoding the cDNAs or genomic clones for the particularantibodies can be used for transformation of a suitable mammalian ornonmammalian host cells. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216,4,912,040, 4,740,461, and 4,959,455 (which patents are herebyincorporated herein by reference). The transformation procedure useddepends upon the host to be transformed. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, particle bombardment, encapsulationof the polynucleotide(s) in liposomes, peptide conjugates, dendrimers,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, NSO₀, HeLa cells, baby hamster kidney(BHK) cells, monkey kidney cells (COS), human hepatocellular carcinomacells (e.g., Hep G2), and a number of other cell lines. Non-mammaliancells including but not limited to bacterial, yeast, insect, and plantscan also be used to express recombinant antibodies. Site directedmutagenesis of the antibody CH2 domain to eliminate glycosylation may bepreferred in order to prevent changes in either the immunogenicity,pharmacokinetic, and/or effector functions resulting from non-humanglycosylation. The expression methods are selected by determining whichsystem generates the highest expression levels and produce antibodieswith constitutive CTLA-4 binding properties.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine sythetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

Antibodies in accordance with the present invention have been analyzedstructurally and functionally. In connection with the structures of theantibodies, amino acid sequences of the heavy and kappa light chainshave been predicted based on cDNA sequences obtained through RT-PCR ofthe hybridomas. See Examples 3 and 4 and FIGS. 1-8. N-terminalsequencing of the antibodies was also conducted in confirmation of theresults discussed in Examples 3 and 4. See Example 5 and FIG. 9. Kineticanalyses of the antibodies were conducted to determine affinities. SeeExample 2. Antibodies in accordance with the invention (and particularlythe 4.1.1, 4.8.1, and 6.1.1 antibodies of the invention) have highaffinities (4.1.1:1.63×10¹⁰ 1/M; 4.8.1:3.54×10¹⁰ 1/M; and 6.1.1:7.2×10⁹1/M). Further, antibodies were analyzed by isoelectric focusing (IEF),reducing gel electrophoresis (SDS-PAGE), size exclusion chromatography,liquid chromatography/mass spectroscopy, and mass spectroscopy andantibody production by the hybridomas was assessed. See Example 6 andFIG. 10.

In connection with functional analysis of antibodies in accordance withthe present invention, such antibodies proved to be potent inhibitors ofCTLA-4 and its binding to its ligands of the B7 family of molecules. Forexample, antibodies in accordance with the present invention weredemonstrated to block CTLA-4 binding to either B7-1 or B7-2. See Example7. Indeed, many of the antibodies in accordance with the inventionpossess nanomolar and subnanomolar IC₅₀s with respect to inhibitingCTLA-4 binding to B7-1 and B7-2. Further, antibodies of the inventionpossess excellent selectivity for CTLA-4 as compared to CD28, CD44,B7-2, or hIgG1. See Example 8. Selectivity is a ratio that reflects thedegree of preferential binding of a molecule with a first agent ascompared to the molecules binding with a second, and optionally othermolecules. Herein, selectivity refers to the degree of preferentialbinding of an antibody of the invention to CTLA-4 as compared to theantibody's binding to other molecules such as CD28, CD44, B7-2, orhIgG1. Selectivity values of antibodies of the invention greater than500:1 are common. Antibodies of the invention have also beendemonstrated to induce or enhance expression of certain cytokines (suchas IL-2 and IFN-γ) by cultured T cells in a T cell blast model. SeeExamples 9 and 10 and FIGS. 12-17. Further, it is expected thatantibodies of the invention will inhibit the growth of tumors inappropriate in vivo tumor models. The design of which models arediscussed in Example 11 and 12.

The results demonstrated in accordance with the present inventionindicate that antibodies of the present invention possess certainqualities that may make the present antibodies more efficacious thancurrent therapeutic antibodies against CTLA-4.

In particular, the 4.1.1, 4.8.1, and 6.1.1 antibodies of the inventionpossess highly desirable properties. Their structural characteristics,functions, or activities provide criteria that facilitate the design orselection of additional antibodies or other molecules as discussedabove. Such criteria include one or more of the following:

Ability to compete for binding to CTLA-4 with one or more of theantibodies of the invention;

Similar binding specificity to CTLA-4 as one or more of the antibodiesof the invention;

A binding affinity for CTLA-4 of about 10⁻⁹ M or greater and preferablyof about 10⁻¹⁰M or greater;

Does not cross react with lower mammalian CTLA-4, including, preferably,mouse, rat, or rabbit and preferably mouse or rat CTLA-4;

Cross reacts with primate CTLA-4, including, preferably, cynomolgous andrhesus CTLA-4;

A selectivity for CTLA-4 over CD28, B7-2, CD44, or hIgG1 of at leastabout 100:1 or greater and preferably of about 300, 400, or 500:1 orgreater;

An IC₅₀ in blocking CTLA-4 binding to B7-2 of about 100 nM or lower andpreferably 5, 4, 3, 2, 1, 0.5, or 0.38 nM or lower;

An IC₅₀ in blocking CTLA-4 binding to B7-1 of about of about 100 nM orlower and preferably 5, 4, 3, 2, 1, 0.5, or 0.50 nM or lower;

An enhancement of cytokine production in one or more in vitro assays,for example:

An enhancement of IL-2 production in a T cell blast/Raji assay of about500 pg/ml or greater and preferably 750, 1000, 1500, 2000, 3000, or 3846pg/ml or greater;

An enhancement of IFN-γ production in a T cell blast/Raji assay of about500 pg/ml or greater and preferably 750, 1000, or 1233 pg/ml or greater;or

An enhancement of IL-2 production in a hPBMC or whole blood superantigenassay of about 500 pg/ml or greater and preferably 750, 1000, 1200, or1511 pg/ml or greater. Expressed another way, it is desirable that IL-2production is enhanced by about 30, 35, 40, 45, 50 percent or morerelative to control in the assay.

It is expected that antibodies (or molecules designed or synthesizedtherefrom) having one or more of these properties will possess similarefficacy to the antibodies described in the present invention.

The desirable functional properties discussed above can often resultfrom binding to and inhibition of CTLA4 by a molecule (i.e., antibody,antibody fragment, peptide, or small molecule) in a similar manner as anantibody of the invention (i.e., binding to the same or similar epitopeof the CTLA4 molecule). The molecule may either be administered directly(i.e., direct administration to a patient of such molecules). Or,alternatively, the molecule may be “administered” indirectly (i.e., apeptide or the like that produces an immune response in a patient(similar to a vaccine) wherein the immune response includes thegeneration of antibodies that bind to the same or similar epitope or anantibody or fragment that is produced in situ after administration ofgenetic materials that encode such antibodies or fragments thereof whichbind to the same or similar epitope). Thus, it will be appreciated thatthe epitope on CTLA4 to which antibodies of the invention bind to can beuseful in connection with the preparation and/or design of therapeuticsin accordance with the invention. In drug design, negative informationis often useful as well (i.e., the fact that an antibody which binds toCTLA4 does not appear to bind to an epitope that acts as an inhibitor ofCTLA4 is useful). Thus, the epitope to which antibodies of the inventionbind that do not lead to the desired functionality can also be veryuseful. Accordingly, also contemplated in accordance with the presentinvention are molecules (and particularly antibodies) that bind to thesame or similar epitopes as antibodies of the invention.

In addition to the fact that antibodies of the invention and theepitopes to which they bind are contemplated in accordance with theinvention, we have conducted some preliminary epitope mapping studies ofcertain antibodies in accordance with the invention and particularly the4.1.1 and the 11.2.1 antibodies of the invention.

As a first step, we conducted BIAcore competition studies to generate arough map of binding as between certain antibodies of the invention inconnection with their ability to compete for binding to CTLA4. To thisend, CTLA4 was bound to a BIAcore chip and a first antibody, undersaturating conditions, was bound thereto and competition of subsequentsecondary antibodies binding to CTLA4 was measured. This techniqueenabled generation of a rough map in to which families of antibodies canbe classified.

Through this process, we determined that the certain antibodies inaccordance with the invention could be categorized as falling into thefollowing epitopic categories:

Category Antibodies Competition for CTLA4 Binding A BO1M* Freelycross-compete with one another; cross- BO2M** compete with category B;some cross- competition with category D B 4.1.1 Freely cross-competewith one another; cross- 4.13.1 compete with category A, C and D. C6.1.1 Freely cross-compete with one another; cross- 3.1.1 compete withcategory B and category D 4.8.1 11.2.1 11.6.1 11.7.1 D 4.14.3Cross-compete with category C and B; some cross-competition withcategory A E 4.9.1 BNI3 blocks 4.9.1 binding to CTLA4 but not BNI3***the reverse * **Available from Biostride. ***Available from Pharmingen.

As a next step, we endeavored to determine if the antibodies of theinvention recognized a linear epitope on CTLA4 under reducing andnon-reducing conditions on Western blots. We observed that none of the4.1.1, 3.1.1, 11.7.1, 11.6.1, or 11.2.1 antibodies of the inventionappeared to recognize a reduced form of CTLA4 on Western blot.Accordingly, it appeared likely that the epitope to which each of theseantibodies bound was not a linear epitope but more likely was aconformational epitope the structure of which may have been abrogatedunder reducing conditions.

Therefore, we sought to determine whether we could learn about residueswithin the CTLA4 molecule that are important for binding of antibodiesof the invention. One manner that we utilized was to conduct kineticassessments of off-rates as between human CTLA4 and two highly conservedprimate CTLA4 molecules (cynomologous and marmoset CTLA4). BIAcorestudies demonstrated that the 4.1.1 antibody of the invention bound tohuman, cynomologous, and marmoset CTLA4 at the same rate. However, withrespect to off-rates (affinity), the 4.1.1 antibody had the highestaffinity (slowest off-rate) for human, a faster off-rate withcynomologous, and a much faster off-rate for marmoset. The 11.2.1antibody of the invention, on the other hand, binds to human,cynomologous, and marmoset CTLA4 at the about the same rate and hasabout the same relative off-rate for each of the three. This informationfurther indicates that the 4.1.1 and 11.2.1 antibodies of the inventionbind to different epitopes on CTLA4.

To further study the epitope to which the category B and C antibodies ofthe invention bind, we conducted certain site directed mutagenesisstudies. Marmoset CTLA4 possesses two important changes at residues 105and 106 relative to human CTLA4. Such differences are a leucine tomethionine change at residue 105 and a glycine to serine change atresidue 106. Accordingly, we mutated cDNA encoding human CTLA4 to encodea mutated CTLA4 having the L105M and G106S changes. The homologuereplacement mutant CTLA4 did not effect binding of a B7.2-IgG1 fusionprotein. Further, binding with the 11.2.1 antibody of the invention wasnot effected. However, such molecule was significantly inhibited in itsability to bind with the 4.1.1 antibody of the invention (similar tomarmoset). Next, we mutated a cDNA encoding marmoset CTLA4 to create amutant marmoset CTLA4 having a S 106G change. Such change resulted inrestoration of stable binding between the 4.1.1 antibody and themarmoset CTLA4 mutant. In addition, we mutated a cDNA encoding marmosetCTLA4 to create a mutant marmoset CTLA4 having a M105L change. Suchchange partially restored binding between the 4.1.1 antibody and themutant CTLA4.

Each of the category B through D antibodies of the invention appear topossess similar functional properties and appear to have the potentialto act as strong anti-CTLA4 therapeutic agents. Further, each of themolecules certain cross-competition in their binding for CTLA4. However,as will be observed from the above discussion, each of the molecules inthe different categories appear to bind to separate conformationalepitopes on CTLA4.

From the foregoing, it will be appreciated that the epitope informationdiscussed above indicates that antibodies (or other molecules, asdiscussed above) that cross-compete with antibodies of the inventionwill likely have certain therapeutic potential in accordance with thepresent invention. Further, it is expected that antibodies (or othermolecules, as discussed above) that cross-compete with antibodies of theinvention (i.e., cross-compete with category B, C and/or D antibodies)will likely have certain additional therapeutic potential in accordancewith the present invention. Additionally, it is expected that antibodies(or other molecules, as discussed above) that cross-compete withantibodies of the invention (i.e., cross-compete with category B, Cand/or D antibodies) and that (i) are not reduced in their binding tomarmoset CTLA4 (similar to the 11.2.1 antibody) or (ii) are reduced intheir binding to marmoset CTLA4 (similar to the 4.1.1 antibody) willlikely have certain additional therapeutic potential in accordance withthe present invention. Antibodies (or other molecules, as discussedabove) that compete with categories A and E may also have certaintherapeutic potential.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Generation of Anti-CTLA-4-Antibody Producing Hybridomas

Antibodies of the invention were prepared, selected, and assayed inaccordance with the present Example.

Antigen Preparation:

Three distinct immunogens were prepared for immunization of theXenoMouse™ mice: (i) a CTLA-4-IgG fusion protein, (ii) a CTLA-4 peptide,and (iii) 300.19 murine lymphoma cells transfected with a mutant ofCTLA-4 (Y201V) that is constitutively expressed on the cell surface.

(i) CTLA-4-IgG1 Fusion Protein:

Expression Vector Construction:

The cDNA encoding the mature extracellular domain of CTLA-4 was PCRamplified from human thymus cDNA library (Clontech) using primersdesigned to published sequence (Eur. J Immunol 18:1901-1905 (1988)). Thefragment was directionally subcloned into pSRS, a Sindbis virusexpression plasmid (InVitrogen), between the human oncostatin M signalpeptide and human IgG gamma 1 (IgG1) CH1/CH2/CH3 domains. The fusionprotein does not contain a hinge domain but contains cysteine 120 in theextracellular domain of CTLA-4 to form a covalent dimer. The resultingvector was called CTLA-4-IgG1/pSR5. The complete CTLA-4-IgG1 cDNA in thevector was sequence confirmed in both strands. The amino acid sequencethe CTLA4-Ig protein is shown below. The mature extracellular domain forCD44 was PCR amplified from human lymphocyte library (Clontech) andsubcloned into pSinRep5 to generate a control protein with the identicalIgG1 tail.

OM-CTLA4-IgG1 Fusion Protein (SEQ ID NO: 100):

MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDLEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPTPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKUnderlined: signal peptideBold: CTLA4 extracellular domain

The cDNAs for mature extracellular domain of CD28 were PCR amplifiedfrom human lymphocyte library (Clontech) and then subcloned into pCDM8(J. Immunol. 151: 5261-71 (1993)) to produce a human IgG1 fusion proteincontaining both thrombin cleavage and hinge regions. Marmoset,Cynomologous, and Rhesus CTLA4 were cloned from mRNA isolated from PHAstimulated PBMCs using standard techniques of degenerate PCR. Sequencingdemonstrated that rhesus and cynomologous amino acid sequence wereidentical with three differences from mature human CTLA4 extracellulardomain (S13N, I17T and L105M). Marmoset demonstrated ten amino aciddifferences from the mature human CTLA4 extracellular domain (V21A,V33I, A41T, A51G, 54I, S71F, Q75K, T88M, L105M and G106S). Site directedmutagenesis was used to make single point mutations of all amino acidsdifferent in marmoset CTLA4 to map amino acids important for interationof the antibodies with human CTLA4-IgG. Mutations of human and marmosetCTLA-IgG for epitope mapping were generated by matchmaker site-directedmutagenesis (Promega). The IgG fusion proteins were produced bytransient transfection of Cos7 cells and purified using standard ProteinA techniques. Mutant CTLA4-IgG proteins were evaluated for binding toantibodies by immunoblotting and using BIAcore analyses.

Recombinant Protein Expression/Purification

Recombinant sindbis virus was generated by electroporating (Gibco) BabyHamster Kidney cells with SP6 in vitro transcribed CTLA-4-IgG1/pSR5 mRNAand DH-26S helper mRNA as described by InVitrogen. Forty eight hourslater recombinant virus was harvested and titered for optimal proteinexpression in Chinese hamster ovary cells (CHO-K1). CHO-K1 cells werecultured in suspension in DMEM/F12 (Gibco) containing 10%heat-inactivated fetal bovine serum (Gibco), non-essential amino acids(Gibco), 4 mM glutamine (Gibco), penicillin/streptomycin (Gibco), 10 mMHepes pH 7.5 (Gibco). To produce CTLA-4-IgG, the CHO-K1 cells wereresuspended at 1×10⁷ cells/ml in DMEM/F12 and incubated with sindbisvirus for one hour at room temperature. Cells were then diluted to1×10⁶/ml in DMEM/F12 containing 1% fetal bovine serum depleted of bovineIgG using protein A sepharose (Pharmacia), non-essential amino acids, 4mM glutamine, 12.5 mM Hepes pH 7.5, and penicillin/streptomycin. Fortyeight hours post-infection cells were pelleted and conditioned media washarvested and supplemented with complete protease inhibitor tablets(Boehringer Mannheim), pH adjusted to 7.5, and filtered 0.2μ (Nalgene).FPLC (Pharmacia) was used to affinity purify the fusion protein using a5 ml protein A HiTrap column (Pharmacia) at a 10 ml/min flow rate. Thecolumn was washed with 30 bed volumes of PBS and eluted with 0.1Mglycine/HCl pH 2.8 at 1 ml/min. Fractions (1 ml) were immediatelyneutralized to pH 7.5 with Tris pH 9. The fractions containingCTLA-4-IgG1 were identified by SDS-PAGE and then concentrated usingcentriplus 50 (Amicon) before applying to sepharose 200 column(Pharmacia) at 1 ml/min using PBS as the solvent. Fractions containingCTLA-4-IgG1 were pooled, sterile filtered 0.2μ (Millipore), aliquotedand frozen at −80° C. CD44-IgG1 was expressed and purified using thesame methods. CD28-IgG was purified from conditioned media fromtransiently transfected Cos7 cells.

Characterization CTLA-4-IgG1:

The purified CTLA-4-IgG1 migrated as a single band on SDS-PAGE usingcolloidal coomassie staining (Novex). Under non-reducing conditionsCTLA-4-IgG1 was a dimer (100 kDa), that reduced to a 50 kDa monomer whentreated with 50 mM DTT Amino acid sequencing of the purified CTLA-4-IgG1in solution confirmed the N-terminus of CTLA-4 (MHVAQPAVVLAS) (SEQ IDNO: 101), and that the oncostatin-M signal peptide was cleaved from themature fusion protein. The CTLA-4-IgG1 bound to immobilized B7.1-IgG ina concentration dependent manner and the binding was blocked by ahamster-anti-human anti-CTLA-4 antibody (BNI3: PharMingen). The sterileCTLA-4-IgG was endotoxin free and quantitated by OD280 using 1.4 as theextinction coefficient. The yield of purified CTLA-4-IgG ranged between0.5-3 mgs/liter of CHO-K1 cells.

(ii) CTLA-4 Peptide:

The following CTLA-4 peptide (SEQ ID NO: 102) was prepared as describedbelow:

NH₂: MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC-CONH₂

ABBREVIATIONS/MATERIALS

NMP, N-Methylpyrrolidinone; TFE, 2,2,2-Trifluoroethanol; DCM,Dichloromethane; FMOC, Fluorenyl Methoxycarbonyl. All reagents weresupplied by Perkin Elmer, with the following exceptions: TFE, AldrichChemical, FMOC-PAL-PEG resin, Perseptive Biosystems. Fmoc-Arg(PMC)-OH,FMOC-Asn(Trt)-OH, FMOC-Asp(tBu)-OH, FMOC-Cys(Trt)-OH, FMOC-Glu(tBu)-OH,FMOC-Gln(Trt)-OH, FMOC-His(Boc)-OH, FMOC-Lys(BOC)-OH, FMOC-Ser(tBu)-OH,FMOC-Thr(tBu)-OH and FMOC-Tyr(tBu)-OH were used for those amino acidsrequiring side chain protecting groups

Peptide Synthesis:

Peptide synthesis was performed on a Perkin-Elmer 431A, retrofitted withfeedback monitoring via UV absorbance at 301 nm (Perkin-Elmer Model 759Adetector). The peptide sequence was assembled on a FMOC-PAL-PEG resinusing conditional double coupling cycles. Forced double couplings wereperformed at cycles 10, 11, 18, 19, 20 and 28 through 33. The resin waswashed with a 50% mixture of DCM and TFE at the completion of eachacylation cycle, followed by capping of unreacted amino groups withacetic anhydride in NMP. Resin was removed from the reactor aftercompleting cycle 49 and the remainder continued to completion. Peptidecleavage from the resin was performed using Reagent K (King et al.International Journal of Protein and Peptide Research 36:255-266 (1990))for 6 hours on 415 mg of resin affording 186 mg crude CTLA-4 peptide.

Peptide Characterization:

25 mg aliquots of the crude CTLA-4 peptide were dissolved in 5 ml 6MGuanidine HCl/100 mM K₂PO₃ at pH6.4 and eluted over a Pharmacia Hi LoadSuperdex 75 16/60 column (16 mm×600 mm, 120 ml bed volume) with 2MGuanidine.HCl/100 mM K₂PO₃ at pH6.4 at 2 ml/min for 180 minutescollecting 5 ml fractions. The fractions were analyzed by loading 1.7 μlof fractions onto a NuPAGE Laemeli gel running with MES running bufferand visualizing via Daichii silver stain protocol. Those fractionsexhibiting a molecular weight of 12 KDa, as judged versus molecularweight standards, were pooled together and stored at 4° C. The combinedfractions were analyzed by UV and gel electrophoresis. Amino acidsequencing was performed by absorbing a 100 microliter sample in aProSorb cartridge (absorbed onto a PVDF membrane) and washing to removethe buffer salts. Sequencing was performed on an Applied Biosystems 420.The expected N-terminal sequence (M H V A Q P A V V L A) was observedImmunoblotting demonstrated that the peptide was recognized by the BNI3anti-human CTLA-4 (PharMingen). To desalt, an aliquot containing 648 μgof material was placed in 3500 Da MWCO dialysis tubing and dialyzedagainst 0.1% TFA/H2O at 4° C. for 9 days with stirring. The entirecontents of the dialysis bag was lyophilyzed to a powder.

(iii) 300.19 Cells Transfected with CTLA-4 (Y201 V)

The full length CTLA-4 cDNA was PCR amplified from human thymus cDNAlibrary (Stratagene) and subcloned into pIRESneo (Clontech). A mutationof CTLA-4 that results in constitutive cell surface expression wasintroduced using MatchMaker Mutagenesis System (Promega). Mutation oftyrosine, Y201 to valine inhibits binding of the adaptin protein AP50that is responsible for the rapid internalization of CTLA-4 (Chuang etal. J. Immunol. 159:144-151 (1997)). Mycoplasma-free 300.19 murinelymphoma cells were cultured in RPMI-1640 containing 10% fetal calfserum, non-essential amino acids, penicillin/streptomycin, 2 mMglutamine, 12.5 mM Hepes pH 7.5, and 25 uM beta-mercaptoethanol. Cellswere electroporated (3×10⁶/0.4 ml serum free RPMI) in a 1 ml chamberwith 20 ug CTLA-4-Y201V/pIRESneo using 200V/1180 uF (Gibco CellPorator).Cells were rested for 10 minutes and then 8 mls of prewarmed completeRPMI media. At 48 hours cells were diluted to 0.5×10⁶/ml in completeRPMI media containing 1 mg/ml G418 (Gibco). Resistant cells wereexpanded and shown to express CTLA-4 on the cell surface using the BNI3antibody conjugated with phycoerythrin (PharMingen). High levelexpressing cells were isolated by sterile sorting.

Immunization and Hybridoma Generation:

XenoMouse mice (8 to 10 weeks old) were immunized (i) subcutaneously atthe base of tails with 1×10⁷ 300.19 cells that were transfected toexpress CTLA-4 as described above, resuspended in phosphate bufferedsaline (PBS) with complete Freund's adjuvant, or (ii) subcutaneously atthe base of tail with (a) 10 μg the CTLA-4 fusion protein or (b) 10 μgCTLA-4 peptide, emulsified with complete Freund's adjuvant. In eachcase, the dose was repeated three or four times in incomplete Freund'sadjuvant. Four days before fusion, the mice received a final injectionof the immunogen or cells in PBS. Spleen and/or lymph node lymphocytesfrom immunized mice were fused with the [murine non-secretory myeloma P3cell line] and were subjected to HAT selection as previously described(Galfre, G. and Milstein, C., “Preparation of monoclonal antibodies:strategies and procedures.” Methods Enzymol. 73:3-46 (1981)). A largepanel of hybridomas all secreting CTLA-4 specific human IgG₂κ or IgG₄κ(as detected below) antibodies were recovered.

Clones 4.1.1 and 11.2.1 were deposited under terms in accordance withthe Budapest Treaty with the American Type Culture Collection (ATCC),University Boulevard. Manassas, Va. 20110-2209 on Jul. 14, 2005.Subclones 4.1.1.1, 4.13.1.1, 6.1.1.3, and 11.2.1.4 were deposited underterms in accordance with the Budapest Treaty with the ATCC on Apr. 29,2003. The clones and subclones have been assigned the following ATCCaccession numbers:

Clone Subclone 4.1.1 PTA-6862 4.1.1.1 PTA-5166 4.13.1.1 PTA-5167 6.1.1.3PTA-5168 11.2.1 PTA 6863 11.2.1.4 PTA-5169

All restrictions imposed by the depositor on the availability to thepublic of the deposited materials will be irrevocably removed upon thegranting of a patent in this application.

ELISA Assay:

ELISA for determination of antigen-specific antibodies in mouse serumand in hybridoma supernatants was carried out as described (Coligan etal., Unit 2.1, “Enzyme-linked immunosorbent assays,” in Currentprotocols in immunology (1994)) using CTLA-4-Ig fusion protein tocapture the antibodies. For animals that are immunized with theCTLA-4-Ig fusion protein, we additionally screen for non-specificreactivity against the human Ig portion of the fusion protein. This isaccomplished using ELISA plates coated with human IgG1 as a negativecontrol for specificity.

In a preferred ELISA assay, the following techniques are used:

ELISA plates are coated with 100 μl/well of the antigen in plate coatingbuffer (0.1 M Carbonate Buffer, pH 9.6 and NaHCO₃ (MW 84) 8.4 g/L).Plates are then incubated at 4° C. overnight. After incubation, coatingbuffer is removed and the plate is blocked with 200 μl/well blockingbuffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1×PBS) andincubated at room temperature for 1 hour. Alternatively, the plates arestored in refrigerator with blocking buffer and plate sealers. Blockingbuffer is removed and 50 μl/well of hybridoma supernatant, serum orother hybridoma supernatant (positive control) and HAT media or blockingbuffer (negative control) is added. The plates are incubated at roomtemperature for 2 hours. After incubation, the plate is washed withwashing buffer (1×PBS). The detecting antibody (i.e., mouse anti-humanIgG2-HRP (SB, #9070-05) for IgG2 antibodies or mouse anti-human IgG4-HRP(SB #9200-05) for IgG4 antibodies) is added at 100 μl/well (mouseanti-human IgG2-HRP @ 1:2000 or mouse anti-human IgG4-HRP @ 1:1000 (eachdiluted in blocking buffer)). The plates are incubated at roomtemperature for 1 hour and then washed with washing buffer. Thereafter,100 μl/well of freshly prepared developing solution (10 ml Substratebuffer, 5 mg OPD (o-phenylenediamine, Sigma Cat No. P-7288), and 10 μl30% H₂O₂ (Sigma)) is added to the wells. The plates are allowed todevelop 10-20 minutes, until negative control wells barely start to showcolor. Thereafter, 100 μl/well of stop solution (2 M H₂SO₄) is added andthe plates are read on an ELISA plate reader at wavelength 490 nm.

Determination of Affinity Constants of Fully Human Mabs by BIAcore:

Affinity measurement of purified human monoclonal antibodies, Fabfragments, or hybridoma supernatants by plasmon resonance was carriedout using the BIAcore 2000 instrument, using general procedures outlinedby the manufacturers.

Kinetic analysis of the antibodies was carried out using antigensimmobilized onto the sensor surface at a low density. Three surfaces ofthe BIAcore sensorchip were immobililized with the CTLA-4-Ig fusionprotein at a density ranging from approximately 390-900 using CTLA-4-Igfusion protein at 20 or 50 μg/ml in 10 mM sodium acetate at pH 5.0 usingthe amine coupling kit supplied by the manufacturer (BIAcore, Inc.). Thefourth surface of the BIAcore sensorchip was immobilized with IgG1 (900RU) and was used as a negative control surface for non-specific binding.Kinetic analysis was performed at a flow rate of 25 or 50 microlitersper minute and dissociation (kd or k_(off)) and association (ka orIc_(on)) rates were determined using the software provided by themanufacturer (BIA evaluation 3.0) that allows for global fittingcalculations.

Example 2 Affinity Measurement of Anti-CTLA-4-Antibodies

In the following Table, affinity measurements for certain of theantibodies selected in this manner are provided:

TABLE I Solid Phase (by BIAcore) Association Dissociation On-ratesOff-rates Constant Constant K_(a) K_(d) KA (1/M) = KD(M) = SurfaceHybrid- (M⁻¹S⁻¹ × (S⁻¹ × k_(a)/k_(d) × K_(d)/k_(a) × Density oma 10⁶)10⁻⁴) 10¹⁰ 10⁻¹⁰ [RU] Moab01 0.68 1.01 0.67 1.48 878.7 0.70 4.66 0.156.68 504.5 0.77 6.49 0.19 8.41 457.2 0.60 3.08 0.20 5.11 397.8 4.1.11.85 0.72 2.58 0.39 878.7 1.88 1.21 1.55 0.64 504.5 1.73 1.54 1.13 0.88457.2 1.86 1.47 1.26 0.79 397.8 4.8.1 0.32 0.07 4.46 0.22 878.7 0.310.23 1.33 0.75 504.5 0.28 0.06 4.82 0.21 397.8 4.14.3 2.81 3.04 0.921.08 878.7 2.88 3.97 0.73 1.38 504.5 2.84 6.66 0.43 2.35 457.2 3.17 5.030.63 1.58 397.8 6.1.1 0.43 0.35 1.21 0.83 878.7 0.46 0.90 0.51 1.98504.5 0.31 0.51 0.61 1.63 457.2 0.45 0.79 0.57 1.76 397.8 3.1.1 1.040.96 1.07 0.93 878.7 0.95 1.72 0.55 1.82 504.5 0.73 1.65 0.44 2.27 457.20.91 2.07 0.44 2.28 397.8 4.9.1 1.55 13.80 0.11 8.94 878.7 1.43 19.000.08 13.20 504.5 1.35 20.50 0.07 15.20 397.8 4.10.2 1.00 2.53 0.39 2.54878.7 0.94 4.30 0.22 4.55 504.5 0.70 5.05 0.14 7.21 457.2 1.00 5.24 0.195.25 397.8 2.1.3 1.24 9.59 0.13 7.72 878.7 1.17 13.10 0.09 11.20 504.51.11 13.00 0.09 11.70 397.8 4.13.1 1.22 5.83 0.21 4.78 878.7 1.29 6.650.19 5.17 504.5 1.23 7.25 0.17 5.88 397.8

As will be observed, antibodies prepared in accordance with theinvention possess high affinities and binding constants.

Example 3

Structures of Anti-CTLA-4-Antibodies Prepared in Accordance with theInvention

In the following discussion, structural information related toantibodies prepared in accordance with the invention is provided.

In order to analyze structures of antibodies produced in accordance withthe invention, we cloned genes encoding the heavy and light chainfragments out of the particular hybridoma. Gene cloning and sequencingwas accomplished as follows:

Poly(A)⁺ mRNA was isolated from approximately 2×10⁵ hybridoma cellsderived from immunized XenoMouse mice using a Fast-Track kit(Invitrogen). The generation of random primed cDNA was followed by PCR.Human V_(H) or human V, family specific variable region primers (Markset al., “Oligonucleotide primers for polymerase chain reactionamplification of human immunoglobulin variable genes and design offamily-specific oligonucleotide probes.” Eur. J. Immunol. 21:985-991(1991)) or a universal human V_(H) primer, MG-30(CAGGTGCAGCTGGAGCAGTCIGG) was used in conjunction with primers specificfor the human Cγ2 constant region (MG-40d;5′-GCTGAGGGAGTAGAGTCCTGAGGA-3′) or Cκ constant region (hκP2; aspreviously described in Green et al., 1994). Sequences of humanMabs-derived heavy and kappa chain transcripts from hybridomas wereobtained by direct sequencing of PCR products generated from poly(A⁺)RNA using the primers described above. PCR products were also clonedinto pCRII using a TA cloning kit (Invitrogen) and both strands weresequenced using Prism dye-terminator sequencing kits and an ABI 377sequencing machine. All sequences were analyzed by alignments to the “VBASE sequence directory” (Tomlinson et al., MRC Centre for ProteinEngineering, Cambridge, UK) using MacVector and Geneworks softwareprograms.

Further, each of the antibodies 4.1.1, 4.8.1, 11.2.1, and 6.1.1 weresubjected to full length DNA sequences. For such sequencing, Poly(A)⁺mRNA was isolated from approximately 4×10⁶ hybridoma cells using mRNADirect kit (Dynal). The mRNA was reverse transcribed using oligo-dT(18)and the Advantage RT/PCR kit (Clonetech). The Variable region database(V Base) was used to design amplification primers beginning at the ATGstart site of the heavy chain DP50 gene(5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGC TG-3′) and to thestop codon of the IgG2 constant region(5′-TTCTCTGATCAGAATTCCTATCATTTACCCGGAGACAGGGAGAGCT-3′). An optimal Kozaksequence (ACCGCCACC) was added 5′ to the ATG start site. The same methodwas used to design a primer to the ATG start site of the kappa chain A27gene (5′-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGAAACCCCAGCGCAG -3′) and the stopcodon of the kappa constant region(5′-TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC-3′). The 012 cDNA wascloned by using a primer to the ATG start site(5′-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGACATGAGGGTCCCCGC T-3) and the kappaconstant region stop codon primer above. The heavy chain cDNAs were alsocloned as genomic constructs by site directed mutagenesis to add an NheIsite at the end of the variable J domain and subcloning an NheI-fragmentcontaining the genomic IgG2 CH1/Hinge/CH2/CH3 regions. The pointmutation to generate NheI site does not alter the amino acid sequencefrom germline. The primer pairs were used to amplify the cDNAs usingAdvantage High Fidelity PCR Kit (Clonetech). Sequence of the PCR wasobtained by direct sequencing using dye-terminator sequencing kits andan ABI sequencing machine. The PCR product was cloned into pEE glutaminesynthetase mammalian expression vectors (Lonza) and three clones weresequenced to confirm somatic mutations. For each clone, the sequence wasverified on both strands in at least three reactions. An aglycosylated4.1.1 antibody was generated by site directed mutagenesis of N294Q inthe CH2 domain. Recombinant antibodies were produced by transienttrasnfection of Cos7 cells in IgG depleted FCS and purified usingstandard Protein A sepharose techniques. Stable transfectants weregenerated by electroporation of murine NSO cells and selection inglutamine free media. Recombinant 4.1.1 with or without glycosylationexhibited identical specificity and affinity for CTLA4 in the in vitroELISA and BIAcore assays.

Gene Utilization Analysis

The following Table sets forth the gene utilization evidenced byselected hybridoma clones of antibodies in accordance with theinvention:

TABLE II Heavy and Light Chain Gene Utilization Heavy Chain Kappa LightChain Clone VH D JH VK JK 4.1.1 DP-50 DIR4 or JH4 A27 JK1 DIR3 4.8.1DP-50 7-27 JH4 A27 JK4 4.14.3 DP-50 7-27 JH4 A27 JK3 6.1.1 DP-50 DIR5 orJH4 A27 JK3 DIR5rc 3.1.1 DP-50 3-3 JH6 012 JK3 4.10.2 DP-50 7-27 JH4 A27JK3 2.1.3 DP-65 1-26 JH6 A10/A26 JK4 4.13.1 DP-50 7-27 JH4 A27 JK311.2.1 DP-50 D1-26 JH6 012 JK3 11.6.1 DP-50 D2-2 or JH6 012 JK3 D411.7.1 DP-50 D3-22 JH4 012 JK3 or D21-9 12.3.1.1 DP-50 D3-3 or JH6 A17JK1 DXP4 12.9.1.1 DP-50 D6-19 JH4 A3/A19 JK4 4.9.1 DP-47 5-24 JH4 L5 JK1and/or 6-19

As will be observed, antibodies in accordance with the present inventionwere generated with a strong bias towards the utilization of the DP-50heavy chain variable region. The DP-50 gene is also referred to as aV_(H) 3-33 family gene. Only one antibody that was selected on the basisof CTLA-4 binding and preliminary in vitro functional assays showed aheavy chain gene utilization other than DP-50. That clone, 2.1.3,utilizes a DP-65 heavy chain variable region and is an IgG4 isotype. TheDP-65 gene is also referred to as a V_(H) 4-31 family gene. On the otherhand, the clone, 4.9.1, which possesses a DP-47 heavy chain variableregion binds to CTLA-4 but does not inhibit binding to B7-1 or B7-2. InXenoMouse mice, there are more than 30 distinct functional heavy chainvariable genes with which to generate antibodies. Bias, therefore, isindicative of a preferred binding motif of the antibody-antigeninteraction with respect to the combined properties of binding to theantigen and functional activity.

Mutation Analysis

As will be appreciated, gene utilization analysis provides only alimited overview of antibody structure. As the B-cells in XenoMouseanimals stocastically generate V-D-J heavy or V-J kappa light chaintranscripts, there are a number of secondary processes that occur,including, without limitation, somatic hypermutation, n-additions, andCDR3 extensions. See, for example, Mendez et al. Nature Genetics15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996. Accordingly, to further examine antibody structurepredicted amino acid sequences of the antibodies were generated from thecDNAs obtained from the clones. In addition, N-terminal amino acidsequences were obtained through protein sequencing.

FIG. 1 provides nucleotide and predicted amino acid sequences of theheavy and kappa light chains of the clones 4.1.1 (FIG. 1A), 4.8.1 (FIG.1B), 4.1-4.3 (FIG. 1C), 6.1.1 (FIG. 1D), 3.1.1 (FIG. 1E), 4.1-0.2 (FIG.1F), 2.1.3 (FIG. 1G), 4.1-3.1 (FIG. 1H), 11.2.1 (FIG. 1I), 11.6.1 (FIG.1J), 11.7.1 (FIG. 1K), 12.3.1.1 (FIG. 1L), and 12.9.1.1 (FIG. 1M). InFIGS. 1A, 1B, and 1D, extended sequences of the antibodies 4.1.1, 4.8.1,and 6.1.1 were obtained by full length cloning of the cDNAs as describedabove. In such Figures, the signal peptide sequence (or the basesencoding the same) are indicated in bold and sequences utilized for the5′ PCR reaction are underlined.

FIG. 2 provides a sequence alignment between the predicted heavy chainamino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1,4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1 and thegermline DP-50 (3-33) amino acid sequence. Differences between the DP-50germline sequence and that of the sequence in the clones are indicatedin bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3sequences of the antibodies as shaded.

FIG. 3 provides a sequence alignment between the predicted heavy chainamino acid sequence of the clone 2.1.3 and the germline DP-65 (4-31)amino acid sequence. Differences between the DP-65 germline sequence andthat of the sequence in the clone are indicated in bold. The Figure alsoshows the positions of the CDR1, CDR2, and CDR3 sequences of theantibody as underlined.

FIG. 4 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1,4.10.2, and 4.13.1 and the germline A27 amino acid sequence. Differencesbetween the A27 germline sequence and that of the sequence in the cloneare indicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibody as underlined. Apparentdeletions in the CDR1s of clones 4.8.1, 4.14.3, and 6.1.1 are indicatedwith “0s”.

FIG. 5 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clones 3.1.1, 11.2.1, 11.6.1, and11.7.1 and the germline 012 amino acid sequence. Differences between the012 germline sequence and that of the sequence in the clone areindicated in bold. The Figure also shows the positions of the CDR1,CDR2, and CDR3 sequences of the antibody as underlined.

FIG. 6 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 2.1.3 and the germline A10/A26amino acid sequence. Differences between the A10/A26 germline sequenceand that of the sequence in the clone are indicated in bold. The Figurealso shows the positions of the CDR1, CDR2, and CDR3 sequences of theantibody as underlined.

FIG. 7 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 12.3.1 and the germline A17 aminoacid sequence. Differences between the A17 germline sequence and that ofthe sequence in the clone are indicated in bold. The Figure also showsthe positions of the CDR1, CDR2, and CDR3 sequences of the antibody asunderlined.

FIG. 8 provides a sequence alignment between the predicted kappa lightchain amino acid sequence of the clone 12.9.1 and the germline A3/A19amino acid sequence. Differences between the A3/A19 germline sequenceand that of the sequence in the clone are indicated in bold. The Figurealso shows the positions of the CDR1, CDR2, and CDR3 sequences of theantibody as underlined.

FIG. 22 provides a series of additional nucleotide and amino acidsequences of the following anti-CTLA-4 antibody chains:

4.1.1:

-   -   full length 4.1.1 heavy chain (cDNA 22(a), genomic 22(b), and        amino acid 22(c));    -   full length aglycosylated 4.1.1 heavy chain (cDNA 22(d) and        amino acid 22(e));    -   4.1.1 light chain (cDNA 22(f) and amino acid 22(g));

4.8.1:

-   -   full length 4.8.1 heavy chain (cDNA 22(h) and amino acid 22(i));    -   4.8.1 light chain (cDNA 22(j) and amino acid 22(k));

6.1.1:

-   -   full length 6.1.1 heavy chain (cDNA 22(l) and amino acid 22(m));    -   6.1.1 light chain (cDNA 22(n) and amino acid 22(o));

11.2.1:

-   -   full length 11.2.1 heavy chain (cDNA 22(p) and amino acid        22(q)); and    -   11.2.1 light chain (cDNA 22 (r) and amino acid 22(s)).

Signal peptide sequences are shown in bold and large text. The openreading frames in the full length 4.1.1 genomic DNA sequence (FIG. 22(b)) are underlined. And, the mutations introduced to make theaglycosylated 4.1.1 heavy chain and the resulting change (N294Q) areshown in double underline and bold text (cDNA (FIG. 22( b) and aminoacid (FIG. 22( c)).

Example 4 Analysis of Heavy and Light Chain Amino Acid Substitutions

In FIG. 2, which provides a sequence alignment between the predictedheavy chain amino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3,6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and12.9.1.1 and the germline DP-50 (3-33) amino acid sequence, aninteresting pattern emerges. In addition to the fact of the bias forheavy chain DP-50 in the majority of the clones, there is relativelylimited hypermutation in the antibodies relative to the germline DP-50gene. For example, clones 3.1.1 and 11.2.1 have no mutations. Moreover,the mutations in the other clones are generally conservative changes,involving substitutions of amino acids with similar properties to theamino acids in the germline. Mutations within many of the CDR1 and CRD2sequences are particularly conservative in nature. Three of the heavychains represented in FIG. 2, 4.10.2, 4.13.1, and 4.14.3, are clearlyderived from a single recombination event (i.e., derive from anidentical germinal center) and are nearly identical in sequence. Ifthese three are considered as a single sequence, then, among the 10different antibodies containing the DP50 heavy chain, in CDR1 and CDR2there are 3 positions in which a nonpolar residue is replaced by anothernonpolar residue, 12 in which a polar uncharged residue is replaced byanother polar uncharged residue, and 1 in which a polar charged residueis replaced by another polar charged residue. Further, there are twopositions in which two residues which are very similar structurally,glycine and alanine, are substituted for one another. The only mutationsnot strictly conservative involve 3 substitutions of a polar chargedresidue for a polar uncharged residue and one substitution of a nonpolarresidue for a polar residue.

The light chains of these antibodies are derived from 5 different Vkgenes. The A27 gene is the most heavily represented and is the source of6 different light chains. Comparison of these 6 sequences reveals twonoteworthy features. First, in three of them, 4.8.1, 4.14.3, and 6.1.1,contain deletions of one or two residues in CDR1, a rare event. Second,there is a strong prejudice against the germline serine at position sixin CDR3 in that the serine has been replaced in every sequence. Thissuggests that a serine at this position is incompatible with CTLA4binding.

It will be appreciated that many of the above-identified amino acidsubstitutions exist in close proximity to or within a CDR. Suchsubstitutions would appear to bear some effect upon the binding of theantibody to the CTLA-4 molecule. Further, such substitutions could havesignificant effect upon the affinity of the antibodies.

Example 5

N-Terminal Amino Acid Sequence Analysis of Antibodies in Accordance withthe Invention

In order to further verify the composition and structure of theantibodies in accordance with the invention identified above, wesequenced certain of the antibodies using a Perkin-Elmer sequencer. Bothheavy and kappa light chains of the antibodies were isolated andpurified through use of preparative gel electrophoresis andelectroblotting techniques and thereafter directly sequenced asdescribed in Example 6. A majority of the heavy the heavy chainsequences were blocked on their amino terminus. Therefore, suchantibodies were first treated with pyroglutamate aminopeptidase andthereafter sequenced.

The results from this experiment are shown in FIG. 9. FIG. 9 alsoprovides the molecular weight of the heavy and light chains asdetermined by mass spectroscopy (MALDI).

Example 6

Additional Characterization of Antibodies in Accordance with theInvention

FIG. 10 provides certain additional characterizing information aboutcertain of the antibodies in accordance with the invention. In theFigure, data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3, and6.1.1 is summarized. The following data is provided: Concentration,isoelectric focusing (IEF), SDS-PAGE, size exclusion chromatography,FACS, mass spectroscopy (MALDI), JO and light chain N-terminalsequences.

Generally, the data was generated as follows:

Materials and Methods

Protein concentration was determined at 280 nm from a UV scan (200-350nm), where 1.58 absorbance units at 280 nm equaled 1 mg/ml.

SDS-PAGE was performed using the Novex NuPAGE electrophoresis systemwith a 10% NuPAGE gel and MES running buffer. Samples were prepared bydiluting 3:1 with 4× NuPAGE sample buffer (+/−) beta-mercaptoethanol,heated and ˜5 ug of protein was loaded onto the gel. The gel was thenstained with Brilliant Blue R staining solution (Sigma cat.# B-6529) andmolecular size estimates were made by comparing stained bands to“Perfect Protein Markers” (Novagen cat#69149-3).

For N-terminal sequencing, samples were run as above on NuPAGE gels,transferred to Pro Blot immobilization membrane (Applied Biosystems)then stained with Coomassie Blue R-250. The stained protein bands wereexcised and subjected to sequence analysis by automated Edmandegradation on an Applied Biosystems 494 Procise HT Sequencer.

Isoelectric focusing (IEF) was performed using Pharmacia IEF 3-9 pHastgels (cat#17-0543-01). Samples were diluted in 10% glycerol to ˜0.8mg/ml and 1 ul was loaded onto gel and then silver stained. The pIestimates were made by comparing stained bands to broad range (pH3-10)IEF standards (Pharmacia cat #17-0471-01)

Size exclusion chromatography (SEC) was carried in phosphate bufferedsaline (PBS) on the Pharmacia SMART system using the Superdex 75 PC3.2/30 column. Molecular size estimates were made by comparing peakretention time to the retention times of gel

For FACS studies, human peripheral T cells were prepared and stimulatedfor 48 hours. T cells were washed once, resuspended in FACS buffer at1×10⁶ cells/100 ul and stained for CD3 surface expression with 10 ul ofanti-CD3-FITC (Immunotech, Marseille, France) for 30 minutes at roomtemperature. Cells were washed twice, then fixed, permeabilized (Fix andPerm, Caltag), and stained for intracellular CTLA-4 expression with 10ul anti-CD152-PE (Pharmingen). Flow cytometry was performed using aBecton Dickinson FACSort. Quadrants were set by analysis of relevantisotype control antibodies (Caltag).

As was discussed above, anti-CTLA-4 antibodies have been demonstrated topossess certain powerful immune modulation activities. The followingexperiments were carried out in order to determine if antibodies inaccordance with the present invention possessed such activities. Ingeneral, the experiments were designed to assess ability of theantibodies to inhibit the interaction between CTLA-4 and B7 molecules,be selective as between CTLA-4 and B7 molecules and CD28, and promote Tcell cytokine production, including, but not limited to IL-2 and/orIFN-γ expression. Further, examination of cross-reactivity of antibodiesof the invention with certain human tissues and CTLA-4 molecules inother species (e.g., mouse and primate) was undertaken.

Example 7

Competition ELISA: Inhibition of CTLA-4/B7-1 or B7-2 Interaction byAntibodies in Accordance with the Invention

An in vitro assay was conducted to determine if antibodies in accordancewith the present invention were capable of inhibiting the binding ofCTLA-4 with either B7-1 or B7-2. As will be appreciated, antibodies ofthe invention that are capable of inhibiting the binding of CTLA-4 withB7 molecules would be expected to be candidates for immune regulationthrough the CTLA-4 pathway. In the assay, the following materials andmethods were utilized:

Materials and Methods

3 nM B7.1-Ig(G1) or B7.2-Ig(G1) (Repligen, Inc. Needham, Mass.) inDulbecco's PBS was coated on 96-well MaxiSorp plates (Nunc, Denmark,#439454) and incubated at 4° C. overnight. On day 2, B7-Ig was removedand plates were blocked with 1% BSA plus 0.05% Tween-20 in D-PBS for twohours. Plates were washed 3× with wash buffer (0.05% Tween-20 in D-PBS).Antibody at appropriate test concentrations and CTLA-4-Ig(G4) (0.3 nMfinal conc.) (Repligen, Inc. Needham, Mass.) were pre-mixed for 15minutes and then added to the B7-Ig coated plate (60 ul total volume)and incubated at RT for 1.5 hours. Plates were washed 3× and 50 μl of a1 to 1000 dilution of HRP-labeled mouse anti-human IgG4 antibody (Zymed,San Francisco, Calif., #05-3820) was added and incubated at RT for 1hour. Plates were washed 3× and 50 μl TMB Microwell peroxidase substrate(Kirkegaard & Perry, Gaithersburg, Md., #50-76-04) was added andincubated at RT for 20 minutes, and then 50 μl 1N H₂SO₄ was added to theplate. Plates were read at 450 nm using a Molecular Devices plate reader(Sunnyvale, Calif.). All samples were tested in duplicate. Maximalsignal was defined as CTLA-4-Ig binding in the absence of test antibody.Non-specific binding was defined as absorbance in the absence ofCTLA-4-Ig and test antibody.

The results from the assay are provided in Table IIIA and IIIB In TableIIIA, results are shown for a variety of antibodies in accordance withthe invention. In Table IIIB, results are shown comparing the 4.1.1antibody of the invention with the 11.2.1 antibody of the invention froma separate experiment.

TABLE IIIA CTLA4/B7.2 CTLA4/B7.1 Clone Comp. ELISA Comp. ELISA CTLA-4-IgIsotype IC50 (nM) IC50 (nM) CT3.1.1 IgG2 0.45 ± 0.07 (n = 3) 0.63 ± 0.10(n = 2) CT4.1.1 IgG2 0.38 ± 0.06 (n = 5) 0.50 ± 0.05 (n = 2) CT4.8.1IgG2 0.57 ± 0.03 (n = 3) 0.17 ± 0.28 (n = 2) CT4.9.1 IgG2Non-competitive non-competitive (n = 3) (n = 2) CT4.10.2 IgG2 1.50 ±0.37 (n = 3) 3.39 ± 0.31 (n = 2) CT4.13.1 IgG2 0.49 ± 0.05 (n = 3) 0.98± 0.11 (n = 2) CT4.14.3 IgG2 0.69 ± 0.11 (n = 3) 1.04 ± 0.15 (n = 2)CT6.1.1 IgG2 0.39 ± 0.06 (n = 3) 0.67 ± 0.07 (n = 2)

TABLE IIIB CTLA4/B7.2 CTLA4/B7.1 Clone Comp. ELISA Comp. ELISA CTLA-4-IgIsotype IC50 (nM) IC50 (nM) CT4.1.1 IgG2 0.55 ± 0.08 (n = 4) 0.87 ± 0.14(n = 2) CT11.2.1 IgG2 0.56 ± 0.05 (n = 4) 0.81 ± 0.24 (n = 2)

Example 8

Selectivity Ratios of Antibodies of the Invention with Respect to CTLA-4Versus Either CD28 or B7-2

Another in vitro assay was conducted to determine the selectivity ofantibodies of the invention with respect to CTLA-4 versus either CD28 orB7-2. The following materials and methods were utilized in connectionwith the experiments:

CTLA-4 Selectivity ELISA: Materials and Methods

A 96-well FluroNUNC plate (Nunc Cat No. 475515) was platecoated withfour antigens: CTLA-4/Ig, CD44/Ig, CD28/Ig, and B7.2/Ig (antigensgenerated in-house). The antigens were platecoated overnight at +4° C.at 1 ug/ml 100 ul/well in 0.1M sodium bicarbonate buffer, pH 9.6. Theplate was then washed with PBST (PBS+0.1% Tween-20) three times using aNUNC plate washer. The plate was blocked with PBST+0.5% BSA at 150ul/well. The plate was incubated at RT for 1 hour then washed with PBSTthree times. Next the anti-CTLA-4 antibodies of the invention werediluted in block at 1 μg/ml and were added to the plate. The plate wasincubated at RT for 1 hour then washed with PBST three times. The wellsthat contained the antibodies of the invention were then treated with100 μl/well anti-human IgG2-HRP (Southern Biotech Cat No. 9070-05) at a1:4000 dilution in block. Also, one row was treated with anti-human IgG(Jackson Cat No. 209-035-088) to normalize for platecoating. Thisantibody was diluted to 1:5000 in block and added at 100 ul/well. Also,one row was treated with anti-human CTLA-4-HRP (Pharmingen Cat No.345815/Custom HRP conjugated) as a positive control. This antibody wasused at 0.05 ug/ml diluted in block. The plate was incubated at RT for 1hour then washed with PBST three times. LBA chemiluminescent substrate(Pierce) was added at 100 μl/well and the plate was incubated on aplateshaker for 5 min. The plate was then read using a lumi-imager for a2 min. exposure.

IGEN CTLA-4-Ig Selectivity Binding Assay: Materials and Methods

M-450 Dynabeads (Dynal A. S, Oslo, Norway #140.02) were washed 3× withNa phosphate buffer, pH 7.4 and resuspended in Na phosphate buffer. 1.0μg CTLA-4-Ig(G1), 1.0 μg CD28-Ig(G1) or 1.0 to 3.0 μg B7.2-Ig(G1)(Repligen, Inc. Needham, Mass.) were added to 100 μl of beads andincubated overnight on a rotator at 4° C. On day 2 the beads were washed3× in 1% BSA plus 0.05% Tween-20 in Dulbecco's PBS and blocked for 30minutes. Beads were diluted 1 to 10 with blocking buffer and 25 μl ofthe coated beads were added to 12×75 mm polypropylene tubes. All sampleswere tested in duplicate. 50 μl test antibody (1 μg/ml finalconcentration) or blocking buffer was added to the tubes and incubatedfor 30 minutes on the Origen 1.5 Analyzer carousel (IGEN International,Inc., Gaithersburg, Md.) at RT, vortexing at 100 rpm. 25 μl ofruthenylated murine anti-human IgG1, IgG2 or IgG4 (Zymed, Inc. SanFrancisco, Calif. #05-3300, 05-3500 and 05-3800) (final concentration of3 μg/ml in 100 μl total volume) was added to the tubes. Tubes wereincubated for 30 minutes at RT on the carousel vortexing at 100 rpm. 200μl of Origen assay buffer (IGEN International, Inc., Gaithersburg, Md.#402-050-03) per tube was added and briefly vortexed and then the tubeswere counted in the Origen Analyzer and ECL (electrochemiluminescence)units were determined for each tube. Normalization factors weredetermined to correct for differences in binding of fusion proteins toDynabeads, and ECL units were corrected for non-specific binding beforecalculating selectivity ratios.

The results from the assays are provided in Tables IVA and IVB.

TABLE IVA CTLA4/CD28 CTLA4/B7.2 CTLA4/CD44 CTLA4/CD28 CTLA4/B7.2 CloneIsotype ELISA ELISA ELISA IGEN IGEN 3.1.1 IgG2 >500:1 (n = 3) >500:1 (n= 3) >500:1 (n = 3) >500:1 (n = 2) >500:1 (n = 1)  195:1 (n = 1) 4.1.1IgG2 >500:1 (n = 3) >500:1 (n = 2) >500:1 (n = 3) >500:1 (n = 1) >500:1(n = 1)  485:1 (n = 1)  261:1 (n = 1)  107:1 (n = 1) 4.8.1 IgG2 >500:1(n = 3) >500:1 (n = 2) >500:1 (n = 3) >500:1 (n = 2) >500:1 (n = 2) 190:1 (n = 1) 4.9.1 IgG2 >500:1 (n = 2) >500:1 (n = 2) >500:1 (n =3) >500:1 (n = 1) >500:1 (n = 1)  244:1 (n = 1)  33:1 (n = 1) 4.10.2IgG2 >500:1 (n = 3) >500:1 (n = 3) >500:1 (n = 3) >500:1 (n = 1) >500:1(n = 1) 4.13.1 IgG2 >500:1 (n = 2) >500:1 (n = 3) >500:1 (n = 3) >500:1(n = 1) >500:1 (n = 2)  46:1 (n = 1)  329:1 (n = 1) 4.14.3 IgG2 >500:1(n = 2) >500:1 (n = 2) >500:1 (n = 2) >413:1 (n = 1) >234:1 (n = 1) 80:1 (n = 1)  10:1 (n = 1)  126:1 (n = 1) 6.1.1 IgG2 >500:1 (n =2) >500:1 (n = 3) >500:1 (n = 3) >500:1 (n = 2) >500:1 (n = 2)  52:1 (n= 1)

TABLE IVB CTLA4/CD28 CTLA4/B7.2 CTLA4/hIgG Clone Isotype ELISA ELISAELISA 4.1.1 IgG2 >500:1 (n = 3) >500:1 (n = 2) >500:1 (n = 3) 11.2.1IgG2 >500:1 (n = 3) >500:1 (n = 3) >500:1 (n = 3)

Example 9 Human T-Cell Signal Model

In order to further define the activity of antibodies in accordance withthe invention to act as immune regulators, we developed certain T-cellassays in order to quantify the enhancement of T-cell IL-2 productionupon blockade of CTLA-4 signal with the antibodies. The followingmaterials and methods were utilized in connection with the experiments:

Materials and Methods

Freshly isolated human T cells were prepared by using Histopaque (Sigma,St. Louis, Mo. #A-70543) and T-kwik (Lympho-Kwik, One Lambda, CanogaPark, Calif., #LK-50-T), and stimulated with PHA (1 μg/ml) (PurifiedPhytohemagglutinin, Murex Diagnostics Ltd. Dartford, England, #HA 16) inmedium (RPMI 1640 containing L-glutamine, MEM non-essential amino acids,penicillin, streptomycin, 25 mM Hepes and 10% FBS) at a concentration of1×10⁶ cells/ml and incubated at 37° C. for 2 days. The cells were washedand diluted in medium to 2×10⁶ cells/ml. Raji cells (Burkitt lymphoma,Human ATCC No.: CCL 86 Class II American Type Culture CollectionRockville, Md.) were treated with mitomycin C (Sigma St. Louis, Mo., #M-4287) (25 μg/ml) for one hour at 37° C. The Raji cells were washed 4×in PBS and resuspended at 2×10⁶ cells/ml. Human T cell blasts(5×10⁵/ml), Raji cells (5×10⁵/ml) and anti-CTLA-4 antibodies or anisotyped-matched control antibody at various concentrations were addedto 96-well microtiter plates and the plates were incubated at 37° C. for72 hours. Total volume per well was 200 μl. Seventy-two hours poststimulation, the plates were spun down and supernatant removed andfrozen for later determination of IL-2 (Quantikine IL-2 ELISA kit, R&DSystems, Minneapolis, Minn., #D2050) and IFN-γ (Quantikine IFN-g ELISAkit, R&D Systems). Cytokine enhancement was defined as the differencebetween cytokine levels in cultures containing an anti-CTLA-4 blockingmAb versus an isotype-matched control antibody. For flow cytometryexperiments, Raji cells were washed 1× with FACS buffer (PBS containing2% heat inactivated FCS, 0.025% sodium azide). Cell pellets wereresuspended in FACS buffer at 1×10⁶ cells/100 μl and incubated with 10μl of anti-CD80-PE (Becton Dickinson, San Jose, Calif.) or anti-CD86-PE(Pharmingen, San Diego, Calif.) for 30 minutes at room temperature.Cells were washed twice and resuspended in 1 ml FACS buffer. Flowcytometry was performed using a Becton Dickinson FACSort. Histogrammarkers were set by analysis of relevant isotype control antibodies(Caltag, Burlingame, Calif.).

In general, we have developed an assay that can be used for rapiddetermination of T-cell IL-2 upregulation. As will be appreciated,stimulation of T cells is B7 and CD28 dependent. Further, washed Tblasts do not make detectable IL-2 and Raji cells do not make detectableIL-2 even when stimulated with LPS or PWM. However, in combination, theT blasts co-cultured with Raji cells can model B7, CTLA-4, and CD28signaling events and the effects of antibodies thereon can be assessed.

FIG. 11 shows the expression of B7-1 and B7-2 on Raji cells usinganti-CD80-PE and anti-CD86-PE mAbs using flow cytometry (FACs) asdescribed in Example 6.

FIG. 12 shows the concentration dependent enhancement of IL-2 productionin the T cell blast/Raji assay induced by CTLA-4 blocking antibodies(BNI3 (PharMingen) and the 4.1.1, 4.8.1, and 6.1.1 antibodies of theinvention).

FIG. 13 shows the concentration dependent enhancement of IFN-γproduction in the T cell blast/Raji assay induced by CTLA-4 blockingantibodies (BNI3 (PharMingen) and the 4.1.1, 4.8.1, and 6.1.1 antibodiesof the invention) (same donor T cells).

FIG. 14 shows the mean enhancement of IL-2 production in T cells from 6donors induced by CTLA-4 blocking antibodies in the T cell blast/Rajiassay. It is interesting to consider that the mAb, CT4.9.1, binds toCTLA4 but does not block B7 binding. Thus, simply binding to CTLA-4 isinsufficient by itself to provide a functional antibody of theinvention.

FIG. 15 shows the mean enhancement of IFN-γ production in T cells from 6donors induced by CTLA-4 blocking antibodies in the T cell blast/Rajiassay.

FIG. 19 shows a comparison between the 4.1.1 and 11.2.1 antibodies ofthe invention at a concentration of 30 μg/ml in the 72 hour T cellblast/Raji assay as described in this Example 9 and the Superantigenassay described in Example 10.

FIG. 20 shows the concentration dependent enhancement of IL-2 productionin the T cell blast/Raji assay induced by the 4.1.1 and 11.2.1 CTLA4antibodies of the invention.

The following Table IVc provides information related to mean enhancementand range of enhancement of cytokine response in the Raji and SEA assaysof the invention. Each of the experiments included in the results arebased on antibody at a dose of 30 μg/ml and measured at 72 hours.Numbers of donors used in the experiments as well as responses areshown.

TABLE IVC Mean Range Enhancement Enhancement Donor Assay mAb Cytokinepg/ml SEM pg/ml n Response T cell blast/Raji 4.1.1 IL-2 3329 408 0 to8861 42 19 of 21 T cell blast/Raji 4.1.1 IFN-γ 3630 980 600 to 13939 1713 of 13 T cell blast/Raji 11.2.1 IL-2 3509 488 369 to 6424 18 14 of 14SEA (PBMC) 4.1.1 IL-2 2800 312 330 to 6699 42 17 of 17 SEA (PBMC) 11.2.1IL-2 2438 366 147 to 8360 25 15 of 15 SEA 4.1.1 IL-2 6089 665 −168 to18417 46 15 of 17 (Whole Blood) SEA 11.2.1 IL-2 6935 700 −111 to 1180325 12 of 14 (Whole Blood)

Example 10 Human T-Cell Signal Model

We developed a second cellular assay in order to quantify theenhancement of T-cell IL-2 upregulation upon blockade of CTLA-4 signalwith the antibodies. The following materials and methods were utilizedin connection with the experiments:

Materials and Methods

Human PBMC were prepared using Accuspin. Microtiter plates wereprecoated with an anti-CD3 antibody (leu4, Becton Dickinson) (60 ng/ml)and incubated for 2 hours at 37° C. hPBMC were added to the wells at200,000 cells per well. Staphylcoccus enterotoxin A (SEA) (Sigma) wasadded to the wells at 100 ng/ml. Antibodies were added to the wells,usually at 30 μg/ml. Cells were then stimulated for 48, 72 or 96 hours.Plates were centrifuged at the desired time-point and supernatants wereremoved from the wells. Thereafter, supernatants were checked for IL-2production using ELISA (R&D Systems).

Results from these experiments are shown in FIGS. 16, 17, and 21. InFIG. 16, induction of IL-2 production in hPBMC from 5 donors wasmeasured 72 hours after stimulation. In FIG. 17, results are shown frommeasurement of whole blood, analyzing the difference in induction ofIL-2 production in the blood of 3 donors as measured at 72 and 96 hoursafter stimulation.

In FIG. 21, the enhancement of IL-2 production in whole blood of 2donors as measured at 72 hours after stimulation.

Example 11 Tumor Animal Model

We have established an animal tumor model for the in vivo analysis ofanti-murine-CTLA-4 antibodies in inhibiting tumor growth. In the model,a murine fibrosarcoma tumor is grown and the animals are treated withanti-murine-CTLA-4 antibodies. The materials and methods forestablishment of the model are provided below:

Materials and Methods

Female A/J mice (6-8 weeks old) were injected subcutaneously on thedorsal side of the neck with 0.2 ml of Sa1N tumor cells (1×10⁶) (Baskar1995). Anti-murine CTLA-4 or an isotype matched control antibody(PharMingen, San Diego, Calif., 200 ug/animal) were injectedintraperitioneally on days 0, 4, 7 and 14 following the injection oftumor cells. Tumor measurements were taken during the course of the 3-4week experiments using a Starrett SPC Plus electronic caliper (Athol,Mass.) and tumor size was expressed as the surface area covered by tumorgrowth (mm²)

FIG. 18 shows the inhibition of tumor growth with an anti-murine CTLA-4antibody in a murine fibrosarcoma tumor model. As shown in FIG. 18,animals treated with anti-CTLA-4 had a reduction in tumor growth ascompared to animals treated with an isotype control antibody.Accordingly, anti-murine CTLA4 mAbs are capable of inhibiting growth ofa fibrosarcoma in a mouse tumor model.

It is expected that antibodies that are cross-reactive with murineCTLA-4 would perform similarly in the model. However, of the antibodiesof the invention that have been checked for cross-reactivity, none arecross-reactive with murine CTLA-4.

Example 12 Tumor Animal Model

In order to further investigate the activity of antibodies in accordancewith the invention, a xenograft SCID mouse model was designed to testthe eradication of established tumors and their derived metastases. Inthe model, SCID mice are provided with grafted human T cells and areimplanted with patient-derived non-small cell lung cell (NSCL) orcolorectal carcinoma (CC) cells. Implantation is made into the gonadalfat pads of SCID mice. The tumors are allowed to grow, and thereafterremoved. The mice develop human-like tumor and liver metastases. Such amodel is described in Bumpers et al J. Surgical Res. 61:282-288 (1996).

It is expected that antibodies of the invention will inhibit growth oftumors formed in such mice.

INCORPORATION BY REFERENCE

Applicants incorporate herein by reference in its entirety the document“0006590030109.TXT,” which is electronically submitted concurrently withthis application. This document was created on Jun. 14, 2013 and has asize of 167,313 bytes.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. In addition, the following references arealso incorporated by reference herein in their entirety, including thereferences cited in such references:

-   Alegre et al. J Immunol 157:4762-70 (1996)-   Allison and Krummel Science 270:932-933 (1995)-   Balzano et al. Int J Cancer Suppl 7:28-32 (1992)-   Blair et al. J Immunol 160:12-5 (1998)-   Blake and Litzi-Davis BioConjugate Chem. 3:510-513 (1992)-   Boussiotis et al. Proc Natl Acad Sci USA 90:11059-63 (1993)-   Bowie et al. Science 253:164 (1991)-   Bruggeman et al. PNAS USA 86:6709-6713 (1989)-   Bruggeman, M. and Neuberger, M. S. in Methods: A companion to    Methods in Enzymology 2:159-165 (Lerner et al. eds. Academic Press    (1991))-   Bruggemann et al., “Human antibody production in transgenic mice:    expression from 100 kb of the human IgH locus.” Eur. J. Immunol.    21:1323-1326 (1991)-   Bruggemann, M. and Neuberger, M. S. “Strategies for expressing human    antibody repertoires in transgenic mice.” Immunology Today    17:391-397 (1996)-   Brunet et al. Nature 328:267-270 (1987)-   Bumpers et al J. Surgical Res. 61:282-288 (1996)-   Capsey et al. Genetically Engineered Human Therapeutic Drugs    (Stockton Press, NY (1988))-   Castan et al. Immunology 90:265-71 (1997)-   Cepero et al. J Exp Med 188:199-204 (1998)-   Chen et al “Immunoglobulin gene rearrangement in B-cell deficient    mice generated by targeted deletion of the J_(H) locus”    International Immunology 5:647-656 (1993)-   Chen et al. Cell 71:1093-1102 (1992)-   Chen et al. Human Gene Therapy 5:595-601 (1994)-   Chiswell and McCafferty TIBTECH 10:80-84 (1992)-   Choi et al. “Transgenic mice containing a human heavy chain    immunoglobulin gene fragment cloned in a yeast artificial    chromosome” Nature Genetics 4:117-123 (1993)-   Chothia & Lesk J. Mol. Biol. 196:901-917 (1987)-   Chothia et al. Nature 342:878-883 (1989)-   Chuang et al. J. Immunol. 159:144-151 (1997)-   Coligan et al., Unit 2.1, “Enzyme-linked immunosorbent assays,” in    Current protocols in immunology (1994)-   Cwirla et al. PNAS USA 87:6378-6382 (1990)-   Dariavach et al. Eur. J. Immunol. 18:1901-1905 (1988)-   Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp.    101-110 (Volume 5, National Biomedical Research Foundation (1972))    and Supplement 2 to this volume, pp. 1-10-   de Boer et al. Eur J Immunol 23:3120-5 (1993)-   Eckstein, Ed., Oxford University Press, Oxford England (1991))-   Evans et al. J. Med. Chem. 30:1229 (1987)-   Fallarino et al. J Exp Med 188:205-10 (1998)-   Fanger et al. Immunol Methods 4:72-81 (1994)-   Fauchere, J. Adv. Drug Res. 15:29 (1986)-   Fishwild et al., “High-avidity human IgGγ monoclonal antibodies from    a novel strain of minilocus transgenic mice.” Nature Biotech.    14:845-851 (1996).-   Freeman et al. J Exp Med 178:2185-92 (1993)-   Freeman et al. J Immunol 161:2708-15 (1998)-   Freeman et al. Science 262:907-9 (1993)-   Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,    N.Y. (1989))-   Galfre, G. and Milstein, C., “Preparation of monoclonal antibodies:    strategies and procedures.” Methods Enzymol. 73:3-46 (1981)-   Gorman et al. P.N.A.S. 79:6777 (1982)-   Green and Jakobovits J. Exp. Med. 188:483-495 (1998)-   Green et al. Nature Genetics 7:13-21 (1994)-   Grosschedl et al. Cell 41:885 (1985)-   Hanes and Plucthau PNAS USA 94:4937-4942 (1997)-   Harding et al. Nature 356:607-609 (1994)-   Harper et al. J Immunol 147:1037-44 (1991)-   Hathcock et al. Science 262:905-7 (1993)-   Hoganboom et al. Immunol. Reviews 130:43-68 (1992)-   Horspool et al. J Immunol 160:2706-14 (1998)-   Houghten et al. Biotechniques 13:412-421 (1992)-   Houghten PNAS USA 82:5131-5135 (1985)-   Hurwitz et al. J Neuroimmunol 73:57-62 (1997)-   Hurwitz et al. Proc Natl Acad Sci USA 95:10067-71 (1998)-   Immunology—A Synthesis (2^(nd) Edition, E. S. Golub and D. R. Gren,    Eds., Sinauer Associates, Sunderland, Mass. (1991))-   Introduction to Protein Structure (C. Branden and J. Tooze, eds.,    Garland Publishing, New York, N.Y. (1991))-   Jakobovits et al., “Germ-line transmission and expression of a    human-derived yeast artificial-chromosome.” Nature 362:255-258    (1993)-   Jakobovits, A. et al., “Analysis of homozygous mutant chimeric mice:    Deletion of the immunoglobulin heavy-chain joining region blocks    B-cell development and antibody production.” Proc. Natl. Acad. Sci.    USA 90:2551-2555 (1993)-   Jakobovits, A., “Humanizing the mouse genome.” Current Biology    4:761-763 (1994)-   Jakobovits, A., “Production of fully human antibodies by transgenic    mice.” Current Opinion in Biotechnology 6:561-566 (1995)-   Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d    edition, Chafner and Longo, eds., Lippincott Raven (1996))-   Kabat et al. (1991) Sequences of Proteins of Immunological Interest,    N.I.H. publication no. 91-3242-   Kabat Sequences of Proteins of Immunological Interest (National    Institutes of Health, Bethesda, Md. (1987 and 1991))-   Kostelny et al. J. Immunol. 148:1547-1553 (1992)-   Krummel and Allison J Exp Med 182:459-65 (1995)-   Krummel et al. Int Immunol 8:519-23 (1996)-   Kuchroo et al. Cell 80:707-18 (1995)-   Kwon et al. PNAS USA 94:8099-103 (1997)-   LaPlanche et al. Nucl. Acids Res. 14:9081 (1986)-   Lenschow et al. Proc Natl Acad Sci USA 90:11054-8 (1993)-   Lenschow et al. Science 257:789-792 (1992)-   Lin et al. J Exp Med 188:199-204 (1998)-   Linsley et al. J Exp Med 176:1595-604 (1992)-   Linsley et al. J. Exp. Med. 174:561-569 (1991)-   Linsley et al. Science 257:792-795 (1992)-   Liu et al. J. Immunol. 139:3521 (1987)-   Liu et al. P.N.A.S. 84:3439 (1987)-   Lonberg et al., “Antigen-specific human antibodies from mice    comprising four distinct genetic modifications.” Nature 368:856-859    (1994).-   Luhder et al. J Exp Med 187:427-32 (1998)-   Marasco Gene Therapy 4:11-15 (1997)-   Markees et al. J Clin Invest 101:2446-55 (1998)-   Marks et al., “Oligonucleotide primers for polymerase chain reaction    amplification of human immunoglobulin variable genes and design of    family-specific oligonucleotide probes.” Eur. J. Immunol. 21:985-991    (1991)-   McCoy et al. J Exp Med 186:183-7 (1997)-   Mendez et al. Nature Genetics 15:146-156 (1997)-   Needleman and Wunsch J. Mol. Biol. 48:443 (1970)-   Okayama et al. Mol. Cell. Bio. 3:280 (1983)-   Parmley and Smith Gene 73:305-318 (1988)-   Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988)-   Perez et al. Immunity 6:411-7 (1997)-   Perrin et al. Immunol Res 14:189-99 (1995)-   Perrin et al. J Immunol 157:1333-6 (1996)-   Pinalla et al. Biotechniques 13:901-905 (1992)-   Proteins, Structures and Molecular Principles (Creighton, Ed., W. H.    Freeman and Company, New York (1984))-   Razi-Wolf et al. Proc Natl Acad Sci USA 90:11182-6 (1993)-   Remington's Pharmaceutical Sciences (15^(th) ed, Mack Publishing    Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug,    Seymour-   Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)-   Russel et al. Nucl. Acids Research 21:1081-1085 (1993)-   Schwartz Cell 71:1065 (1992)-   Scott TIBS 17:241-245 (1992)-   Smith and Waterman Adv. Appl. Math. 2:482 (1981)-   Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990)-   Stec et al. J. Am. Chem. Soc. 106:6077 (1984)-   Stein et al. Nucl. Acids Res. 16:3209 (1988)-   Taylor et al., “A transgenic mouse that expresses a diversity of    human sequence heavy and light chain immunoglobulins.” Nucleic Acids    Research 20:6287-6295 (1992)-   Taylor et al., “Human immunoglobulin transgenes undergo    rearrangement, somatic mutation and class switching in mice that    lack endogenous IgM.” International Immunology 6:579-591 (1994)-   The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed.,    McGraw-Hill, San Francisco (1985))-   Thornton et at. Nature 354:105 (1991)-   Tivol et al. Immunity 3:541-7 (1995)-   Townsend and Allison Science 259:368 (1993)-   Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992)-   Tuaillon et al. “Analysis of direct and inverted DJ_(H)    rearrangements in a human Ig heavy chain transgenic minilocus”J.    Immunol. 154:6453-6465 (1995)-   Tuaillon et al., “Human immunoglobulin heavy-chain minilocus    recombination in transgenic mice: gene-segment use in μ and γ    transcripts.” Proc. Natl. Acad. Sci. USA 90:3720-3724 (1993)-   Uhlmann and Peyman Chemical Reviews 90:543 (1990)-   Van Parijs et al. J Exp Med 186:1119-28 (1997)-   Veber and Freidinger TINS p. 392 (1985)-   Vitetta Immunol Today 14:252 (1993)-   Walunas et al. Immunity 1:405-13 (1994)-   Walunas et al. J Exp Med 183:2541-50 (1996)-   Waterhouse et al. Science 270:985-988 (1995)-   Winter and Harris Immunol Today 14:43-46 (1993)-   Wright et al. Crit. Reviews in Immunol. 12125-168 (1992)-   Yang et al. Cancer Res 57:4036-41 (1997)-   Yi-qun et al. Int Immunol 8:37-44 (1996)-   Zon et al. Anti-Cancer Drug Design 6:539 (1991)-   Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp.    87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England    (1991))-   Fry et al. “Specific, irreversible inactivation of the epidermal    growth factor receptor and erbB2, by a new class of tyrosine kinase    inhibitor” Proc Natl Acad Sci USA 95:12022-7 (1998)-   Hoffman et al. “A model of Cdc25 phosphatase catalytic domain and    Cdk-interaction surface based on the presence of a rhodanese    homology domain” J Mol Biol 282:195-208 (1998)-   Ginalski et al. “Modelling of active forms of protein kinases: p38—a    case study” Acta Biochim Pol 44:557-64 (1997)-   Jouko et al. “Identification of csk tyrosine phosphorylation sites    and a tyrosine residue important for kinase domain structure”    Biochem J 322:927-35 (1997)-   Singh et al. “Structure-based design of a potent, selective, and    irreversible inhibitor of the catalytic domain of the erbB receptor    subfamily of protein tyrosine kinases” J Med Chem 40:1130-5 (1997)-   Mandel et al. “ABGEN: a knowledge-based automated approach for    antibody structure modeling” Nat Biotechnol 14:323-8 (1996)-   Monfardini et al. “Rational design, analysis, and potential utility    of GM-CSF antagonists” Proc Assoc Am Physicians 108:420-31 (1996)-   Furet et al. “Modelling study of protein kinase inhibitors: binding    mode of staurosporine and origin of the selectivity of CGP 52411” J    Comput Aided Mol Des 9:465-72 (1995)-   Ill et al. “Design and construction of a hybrid immunoglobulin    domain with properties of both heavy and light chain variable    regions” Protein Eng 10:949-57 (1997)-   Martin et al. “The affinity-selection of a minibody polypeptide    inhibitor of human interleukin-6” EMBO J. 13:5303-9 (1994)-   U.S. patent application Ser. No. 07/466,008, filed Jan. 12, 1990-   U.S. patent application Ser. No. 07/574,748, filed Aug. 29, 1990-   U.S. patent application Ser. No. 07/575,962, filed Aug. 31, 1990-   U.S. patent application Ser. No. 07/610,515, filed Nov. 8, 1990-   U.S. patent application Ser. No. 07/810,279, filed Dec. 17, 1991-   U.S. patent application Ser. No. 07/853,408, filed Mar. 18, 1992-   U.S. patent application Ser. No. 07/904,068, filed Jun. 23, 1992-   U.S. patent application Ser. No. 07/919,297, filed Jul. 24, 1992-   U.S. patent application Ser. No. 07/922,649, filed Jul. 30, 1992-   U.S. patent application Ser. No. 07/990,860, filed Dec. 16, 1992-   U.S. patent application Ser. No. 08/031,801, filed Mar. 15, 1993-   U.S. patent application Ser. No. 08/053,131, filed Apr. 26, 1993-   U.S. patent application Ser. No. 08/096,762, filed Jul. 22, 1993-   U.S. patent application Ser. No. 08/112,848, filed Aug. 27, 1993-   U.S. patent application Ser. No. 08/155,301, filed Nov. 18, 1993-   U.S. patent application Ser. No. 08/161,739, filed Dec. 3, 1993-   U.S. patent application Ser. No. 08/165,699, filed Dec. 10, 1993-   U.S. patent application Ser. No. 08/209,741, filed Mar. 9, 1994-   U.S. patent application Ser. No. 08/234,145, filed Apr. 28, 1994-   U.S. patent application Ser. No. 08/724,752, filed Oct. 2, 1996-   U.S. patent application Ser. No. 08/730,639, filed Oct. 11, 1996-   U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996-   U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996-   U.S. Pat. No. 4,399,216-   U.S. Pat. No. 4,681,581-   U.S. Pat. No. 4,683,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,735,210-   U.S. Pat. No. 4,740,461-   U.S. Pat. No. 4,816,397-   U.S. Pat. No. 4,912,040-   U.S. Pat. No. 4,959,455-   U.S. Pat. No. 5,101,827-   U.S. Pat. No. 5,102,990 (RE 35,500)-   U.S. Pat. No. 5,151,510-   U.S. Pat. No. 5,194,594-   U.S. Pat. No. 5,434,131-   U.S. Pat. No. 5,530,101-   U.S. Pat. No. 5,545,806-   U.S. Pat. No. 5,545,807-   U.S. Pat. No. 5,585,089-   U.S. Pat. No. 5,591,669-   U.S. Pat. No. 5,612,205-   U.S. Pat. No. 5,625,126-   U.S. Pat. No. 5,625,825-   U.S. Pat. No. 5,633,425-   U.S. Pat. No. 5,643,763-   U.S. Pat. No. 5,648,471-   U.S. Pat. No. 5,661,016-   U.S. Pat. No. 5,693,761-   U.S. Pat. No. 5,693,792-   U.S. Pat. No. 5,697,902-   U.S. Pat. No. 5,703,057-   U.S. Pat. No. 5,714,350-   U.S. Pat. No. 5,721,367-   U.S. Pat. No. 5,733,743-   U.S. Pat. No. 5,770,197-   U.S. Pat. No. 5,770,429-   U.S. Pat. No. 5,773,253-   U.S. Pat. No. 5,777,085-   U.S. Pat. No. 5,789,215-   U.S. Pat. No. 5,789,650-   U.S. Pat. No. 5,811,097-   European Patent No. EP 0 546 073 B1-   European Patent No. EP 0 463 151 B1, grant published Jun. 12, 1996-   International Patent Application No. WO 92/02190-   International Patent Application No. WO 92/03918-   International Patent Application No. WO 92/22645-   International Patent Application No. WO 92/22647-   International Patent Application No. WO 92/22670-   International Patent Application No. WO 93/00431-   International Patent Application No. WO 93/12227-   International Patent Application No. WO 94/00569-   International Patent Application No. WO 94/02602, published Feb. 3,    1994-   International Patent Application No. WO 94/25585-   International Patent Application No. WO 94/29444-   International Patent Application No. WO 95/01994-   International Patent Application No. WO 95/03408-   International Patent Application No. WO 95/24217-   International Patent Application No. WO 95/33770-   International Patent Application No. WO 96/14436-   International Patent Application No. WO 96/34096, published Oct. 31,    1996-   International Patent Application No. WO 97/13852-   International Patent Application No. WO 97/20574-   International Patent Application No. WO 97/38137-   International Patent Application No. WO 98/24884-   International Patent Application No. WO 98/24893, published Jun. 11,    1998

EQUIVALENTS

The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

What we claim is:
 1. A monoclonal antibody, or an antigen-bindingportion thereof, that is capable of binding to CTLA-4.
 2. A monoclonalantibody, or an antigen-binding portion thereof, that is capable ofbinding to CTLA-4, wherein said antibody comprises a heavy chainvariable region amino acid sequence that comprises a contiguous aminoacid sequence from within an FR1 sequence through an FR3 sequence thatis encoded by a human V_(H)3-33 family gene and that comprises at leastone of the amino acid substitutions in the CDR1 sequences, CDR2sequences, or framework sequences shown in FIG.
 2. 3. A monoclonalantibody, or an antigen-binding portion thereof, that has one or more ofthe following properties: (a) competes for binding to CTLA-4 with anantibody selected from the group consisting of 2.1.3, 3.1.1, 4.1.1,4.8.1, 4.9.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1,12.3.1.1, and 12.9.1.1; (b) cross-competes for binding to CTLA-4 with anantibody selected from the group consisting of 2.1.3, 3.1.1, 4.1.1,4.8.1, 4.9.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1,12.3.1.1, and 12.9.1.1; and (c) binds to the same epitope of CTLA-4 asan antibody selected from the group consisting of 2.1.3, 3.1.1, 4.1.1,4.8.1, 4.9.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1,12.3.1.1, and 12.9.1.1.
 4. The antibody or antigen-binding portionaccording to claim 1 that is an Fab fragment, an F(ab′)₂ fragment, an F,fragment, a single chain antibody, or a bispecific antibody.
 5. Theantibody according to claim 1 that is an immunoglobulin G (IgG), an IgM,an IgE, an IgA or an IgD molecule.
 6. A pharmaceutical compositioncomprising the antibody or antigen-binding portion according to claim 1and a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising the antibody or antigen-binding portion accordingto claim 3 and a pharmaceutically acceptable carrier.
 8. An isolatedcell line that produces the antibody or antigen-binding portionaccording to claim 1, or the heavy or light chain of said antibody orthe heavy chain antigen-binding portion or light chain antigen-bindingportion of said portion.
 9. A method for producing an anti-CTLA-4antibody or an antigen-binding portion thereof, comprising culturing thecell line according to claim 8 under suitable conditions and recoveringsaid antibody or antigen-binding portion.
 10. An isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the heavy chainor an antigen-binding portion thereof, the light chain or anantigen-binding portion thereof, or both, of the antibody orantigen-binding portion according to claim
 1. 11. A vector comprising anucleotide sequence encoding the heavy chain or an antigen-bindingportion thereof, a nucleotide sequence encoding the light chain or anantigen-binding portion thereof, or both, of the antibody orantigen-binding portion according to claim 1, wherein the vectoroptionally comprises an expression control sequence operably linked tothe nucleotide sequence(s).
 12. A host cell comprising a nucleotidesequence encoding the heavy chain or an antigen-binding portion thereof,a nucleotide sequence encoding the light chain or an antigen-bindingportion thereof, or both, of the antibody or antigen-binding portionaccording to claim
 1. 13. A method for treating a subject in needthereof with an antibody or antigen-binding portion that specificallybinds to CTLA-4, comprising the steps of: (a) administering to a cell ofthe subject an effective amount of a nucleotide sequence encoding theheavy chain or an antigen-binding portion thereof, a nucleotide sequenceencoding the light chain or an antigen-binding portion thereof, or both,of the antibody or antigen-binding portion according to claim 1; and (b)expressing said nucleic acid molecule in the cell.
 14. A method fortreating a subject in need thereof with an antibody or antigen-bindingportion that specifically binds to CTLA-4, comprising the steps of: (a)administering to a cell of the subject an effective amount of anucleotide sequence encoding the heavy chain or an antigen-bindingportion thereof, a nucleotide sequence encoding the light chain or anantigen-binding portion thereof, or both, of the antibody orantigen-binding portion according to claim 3; and (b) expressing saidnucleic acid molecule in the cell.
 15. A non-human transgenic animal ortransgenic plant comprising a nucleotide sequence encoding the heavychain or an antigen-binding portion thereof, a nucleotide sequenceencoding the light chain or an antigen-binding portion thereof, or both,of the antibody or antigen-binding portion according to claim 1, whereinsaid animal or said plant expresses said nucleotide sequence(s).
 16. Amethod for isolating an antibody or an antigen-binding portion thereofthat specifically binds to CTLA-4, comprising the step of isolating theantibody or portion from the non-human transgenic animal or transgenicplant according to claim
 15. 17. A method for treating cancer in asubject, comprising the step of administering to the subject an antibodyor antigen-binding portion according to claim
 1. 18. A method fortreating a tumor in a subject, comprising the step of administering tothe subject a combination comprising an antibody or antigen-bindingportion according to claim 1 and cells expressing granulocyte-macrophagecolony stimulating factor.
 19. A method for treating cancer in asubject, comprising the step of administering to the subject an antibodyor antigen-binding portion according to claim
 3. 20. A method fortreating a tumor in a subject, comprising the step of administering tothe subject a combination comprising an antibody or antigen-bindingportion according to claim 3 and cells expressing granulocyte-macrophagecolony stimulating factor.